Novel compounds and compositions for targeting cancer stem cells

ABSTRACT

The invention provides naphthofuran compounds, polymorphs of naphthofuran compounds, naphthofuran compounds in particle form, purified compositions that contain one or more naphthofuran compounds, purified compositions that contain one or more naphthofuran compounds in particle form, methods of producing these naphthofuran compounds, polymorphs, purified compositions and/or particle forms, and methods of using these naphthofuran compounds, polymorphs, purified compositions and/or particle forms to treat subjects in need thereof.

RELATED APPLICATIONS

This application is a continuation of pending U.S. application Ser. No.13/634,676, filed Oct. 16, 2012, which application is a national stageapplication, filed under 35 U.S.C. §371, of International ApplicationNo. PCT/US2011/029281, filed Mar. 21, 2011, which claims the benefit ofU.S. Provisional Application No. 61/315,886, filed Mar. 19, 2010; U.S.Provisional Application No. 61/315,890, filed Mar. 19, 2010 and U.S.Provisional Application No. 61/325,814, filed Apr. 19, 2010. Thecontents of each of these applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention provides naphthofuran compounds, polymorphs ofnaphthofuran compounds, naphthofuran compounds in particle form,purified compositions that contain one or more naphthofuran compounds,purified compositions that contain one or more naphthofuran compounds inparticle form, methods of producing these naphthofuran compounds,polymorphs, purified compositions and/or particle forms, and methods ofusing these naphthofuran compounds, polymorphs, purified compositionsand/or particle forms to treat subjects in need thereof.

BACKGROUND OF THE INVENTION

Cancer fatalities in the United States alone number in the hundreds ofthousands each year. Despite advances in the treatment of certain formsof cancer through surgery, radiotherapy, and chemotherapy, many types ofcancer are essentially incurable. Even when an effective treatment isavailable for a particular cancer, the side effects of such treatmentcan be severe and result in a significant decrease in quality of life.

Most conventional chemotherapy agents have toxicity and limitedefficacy, particularly for patients with advanced solid tumors.Chemotherapeutic agents cause damage to non-cancerous as well ascancerous cells. The therapeutic index of such compounds (a measure ofthe ability of the therapy to discriminate between cancerous and normalcells) can be quite low. Frequently, a dose of a chemotherapy drug thatis effective to kill cancer cells will also kill normal cells,especially those normal cells (such as epithelial cells) which undergofrequent cell division. When normal cells are affected by the therapy,side effects such as hair loss, suppression of hematopoesis, and nauseacan occur. Depending on the general health of a patient, such sideeffects can preclude the administration of chemotherapy, or, at least,be extremely unpleasant and uncomfortable for the patient and severelydecrease quality of the remaining life of cancer patients. Even forcancer patients who respond to chemotherapy with tumor regression, suchtumor response often is not accompanied by prolongation ofprogression-free survival (PFS) or prolongation of overall survival(OS). As a matter of fact, cancer often quickly progress and form moremetastasis after initial response to chemotherapy. Such recurrentcancers become highly resistant or refractory to chemotherapeutics. Suchrapid recurrence and refractoriness, after chemotherapy, are consideredto be caused by cancer stem cells.

Recent studies have uncovered the presence of cancer stem cells (CSC,also called tumor initiating cells or cancer stem-like cells) which haveself-renewal capability and are considered to be fundamentallyresponsible for malignant growth, relapse and metastasis. Importantly,CSCs are inherently resistant to conventional therapies. Therefore, atargeted agent with activity against cancer stem cells holds a greatpromise for cancer patients (J Clin Oncol. 2008 Jun. 10; 26(17)).Therefore, conventional chemotherapies can kill the bulk of cancercells, but leave behind cancer stem cells. Cancer stem cells can growfaster after reduction of non-stem regular cancer cells by chemotherapy,which is consider the mechanism for the quick relapse afterchemotherapies.

STAT3 is an oncogene which is activated in response to cytokines and/orgrowth factors to promote proliferation, survival, and other biologicalprocesses. STAT3 is activated by phosphorylation of a critical tyrosineresidue mediated by growth factor receptor tyrosine kinases, Januskinases, or the Src family kinases. Upon tyrosine phosphorylation, STAT3forms homo-dimers and translocates to the nucleus, binds to specificDNA-response elements in target gene promoters, and induces geneexpression. STAT3 activates genes involved in tumorigenesis, invasion,and metastasis, including Bcl-xl, Akt, c-Myc, cyclin DE VEGF, andsurvivin. STAT3 is aberrantly active in a wide variety of human cancers,including all the major carcinomas as well as some hematologic tumors.Persistently active STAT3 occurs in more than half of breast and lungcancers, colorectal cancers, ovarian cancers, hepatocellular carcinomas,and multiple myelomas, etc; and more than 95% of head/neck cancers.STAT3 is considered to be one of the major mechanism for drug resistanceof cancer cells. However, STAT3 has proven a difficult target fordiscovering pharmaceutical inhibitor. So far, no direct inhibitor ofSTAT3 with clinically-relevant potency has been identified after decadesof efforts in the industry.

Accordingly, there exists a need for discovering compounds andpharmaceutical compositions for selectively targeting cancer cells, fortargeting cancer stem cells, and for inhibiting STAT3, and methods ofpreparing these compounds and pharmaceutical compositions for clinicalapplications.

The references cited herein are not admitted to be prior art to theclaimed invention.

SUMMARY

The invention provides naphthofuran compounds, polymorphs ofnaphthofuran compounds, purified compositions that contain one or morenaphthofuran compounds, and naphthofuran compounds in particle form.These naphthofuran compounds (including those in particle form),polymorphs, and purified compositions are selective inhibitors of cancerstem cells and STAT3. WO 2009/036099 and WO 2009/036101 disclose thatnaphthofuran compounds target cancer stem cells. It also inhibitsnon-stem cancer cells through inhibiting STAT3. Those compounds arecapable of killing many different types of cancer cells, without causingdamage to normal cells under certain exposure conditions. The compoundscan therefore be used for cancer treatment, especially for the treatmentand prevention of refractory, recurrent, metastatic cancers, orSTAT3-expressing cancers. The publications also describe the processesfor preparing naphthofuran compounds, derivatives, and intermediatesthereof, and the pharmaceutical composition of relevant compounds.

These naphthofuran compounds (including those in particle form),polymorphs, and purified compositions described herein are useful in avariety of indications, including, for example, treating, delaying theprogression of, preventing a relapse of, or alleviating a symptom of acell proliferation disorder. For example, the naphthofuran compounds(including those in particle form), polymorphs, and purifiedcompositions are useful in treating, delaying the progression of,preventing a relapse of, alleviating a symptom of, or otherwiseameliorating a cancer.

In some embodiments, the naphthofuran compound is a polymorph of thecompound shown below, referred to herein as “Compound 1,”

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1.X-ray powder diffraction analysis shown in FIG. 1 was performed using aPhilips PW1800 diffractometer using Cu radiation at 40 KV/30 mA over therange of 5° to 70° with a step size of 0.03° and a counting time of 3hours. Analysis was performed from 2-45° 2-theta using the followingconditions: divergence slit: 0.6 mm, anti-scatter slit: 0.6 mm,receiving slit: 0.1 mm, detector slit: 0.6 mm, step size: 0.02°, steptime: 5 seconds. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.X-ray powder diffraction analysis shown in FIGS. 2 and 3 was performedusing a Bruker D8 Advance diffractometer. Analysis was performed from2-45° 2-theta using the following conditions: divergence slit: 0.6 mm,anti-scatter slit: 0.6 mm, receiving slit: 0.1 mm, detector slit: 0.6mm, step size: 0.02°, step time: 5 seconds.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

The present invention also provides naphthofuran compounds in particleform. For example, the naphthofuran compound in particle form is aparticle of a compound of Formula I shown below, which is active, i.e.,has an efficacy and/or an antitumor activity in vivo. The efficaciousparticle or particles have a defined requirement for particle size, forexample, has a diameter of less than or equal to about 200 μm, about 150μm, about 100 μm, about 40 μm, or about 20 μm, about 10 μm, about 5 μm,about 4 μm, about 3 μm, about 2 μm, about 1 μm, about 0.5 μm, or about0.2 μm. The particle or particles that are larger than the definedparticle size are either inactive or less active.

In some embodiments, the naphthofuran compound in particle form is aparticle of a compound according to Formula I or a salt or solvatethereof,

wherein the particle has a diameter of less than or equal to about 200μm; wherein each (R₁) is independently selected from the groupconsisting of hydrogen, halogen, fluorine, cyano, nitro, CF₃, OCF₃,alkyl, methyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heterocycle, substituted heterocycle, aryl,substituted aryl, OR_(a), SR_(a), and NH₂; wherein n is 4; wherein R₃ isselected from the group consisting of hydrogen, halogen, fluorine,cyano, CF₃, OCF₃, alkyl, methyl, substituted alkyl, halogen-substitutedalkyl, hydroxyl-substituted alkyl, amine-substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heterocycle, substituted heterocycle, aryl, substituted aryl, OR_(a),SR_(a), and NR_(b)R_(c); wherein R_(a) is/are independently selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heterocycle, substituted heterocycle, aryl, and substituted aryl; andwherein R_(b) and R_(c) are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, heterocycle, substituted heterocycle, aryl, andsubstituted aryl, or R_(b) and R_(c) together with the N to which theyare bonded form a heterocycle or substituted heterocycle.

In some embodiments, each (R₁) is independently selected from the groupconsisting of hydrogen, methyl, F (fluorine), Cl, Br, I, OH, and NH₂; R₃is selected from the group consisting of methyl and C(R₈)₃, and each(R₈) is independently selected from the group consisting of hydrogen,methyl, F (fluorine), Cl, Br, I, OH, and NH₂. In some embodiments, atmost two of (R₁) and (R₈) are F (fluorine) with the remainder beinghydrogen. In some embodiments, R₃ is methyl. In a further embodiment,the compound is selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, an enantiomer, diastereomer,tautomer, and a salt or solvate thereof.

In some embodiments, the naphthofuran compound in particle form is aparticle of Compound 1.

In some embodiments, the naphthofuran compound in particle form is aparticle of a polymorph of Compound 1. For example, in some embodiments,the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

In some embodiments, the particle has a diameter of less than or equalto about 160 μm, about 150 μm, about 120 μm, about 100 μm, about 50 μm,about 40 μm, or about 20 μm. In a further embodiment, the particle has adiameter of less than or equal to about 10 μm, about 5 μm, about 4 μm,about 3 μm, about 2 μm, about 1 μm, about 0.5 μm, about 0.2 μm, or about0.1 μm.

The present invention provides a particle or particles of a naphthofurancompound, for example, a compound of Formula I, which are active, i.e.,have an efficacy and/or an antitumor activity. The active particle orparticles have certain size, for example, has a diameter of less than orequal to about 200 μm, about 150 μm, about 100 μm, about 40 μm, or about20 μm, about 10 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm,about 1 μm, about 0.5 μm, about 0.2 μm, or about 0.1 μm. The particle orparticles that are larger than the certain size are either inactive orless active than the particles described herein.

In some embodiments according to the invention, a pharmaceuticalcomposition includes particles of a compound, for example, anaphthofuran, according to Formula I or a salt or solvate thereof. Forexample, in some embodiments, a pharmaceutical composition includesparticles of Compound 1. For example, in some embodiments, apharmaceutical composition includes particles of a polymorph ofCompound 1. For example, in some embodiments, the polymorph is apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 1. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

A fraction of the cumulative total of the particles can have a diameterof less than or equal to about 200 μm. In some embodiments, a fractionof a set of particles can be at least about 1%, at least about 5%, atleast about 10%, at least about 20%, or at least about 30% of the totalnumber of particles in the set. In some embodiments, the fraction is asubstantial fraction. For example, a “substantial fraction” of a set ofparticles can be at least about 99%, at least about 95%, at least about90%, at least about 85%, at least about 80%, at least about 75%, atleast about 70%, at least about 60%, or at least about 50% of the totalnumber of particles in the set. Each (R₁) can be independently selectedfrom the group consisting of hydrogen, halogen, fluorine, cyano, nitro,CF₃, OCF₃, alkyl, methyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocycle,substituted heterocycle, aryl, substituted aryl, OR_(a), SR_(a), andNH₂. n can be a positive integer; for example, n can be 4. R₃ can beselected from the group consisting of hydrogen, halogen, fluorine,cyano, CF₃, OCF₃, alkyl, methyl, substituted alkyl, halogen-substitutedalkyl, hydroxyl-substituted alkyl, amine-substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heterocycle, substituted heterocycle, aryl, substituted aryl, OR_(a),SR_(a), and NR_(b)R_(c). The R_(a) can be independently selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heterocycle, substituted heterocycle, aryl, and substituted aryl. R_(b)and R_(c) can be independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,heterocycle, substituted heterocycle, aryl, and substituted aryl, orR_(b) and R_(c) together with the N to which they are bonded form aheterocycle or substituted heterocycle.

In some embodiments according to the invention, each (R₁) can beindependently selected from the group consisting of hydrogen, methyl, F(fluorine), Cl, Br, I, OH, and NH₂. R₃ can be selected from the groupconsisting of methyl and C(R₈)₃. Each (R₈) can be independently selectedfrom the group consisting of hydrogen, methyl, F (fluorine), Cl, Br, I,OH, and NH₂. In some embodiments, at most two of (R₁) and R₈ can be F(fluorine) with the remainder being hydrogen.

In some embodiments according to the invention, a compound according toFormula I is selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione, and2-ethyl-naphtho[2,3-b]furan-4,9-dione. In some embodiments, a compoundaccording to Formula I is Compound 1. In some embodiments, a compoundaccording to Formula I is a polymorph of Compound 1. For example, insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

For example, the pharmaceutical composition can have at least about 90%of the cumulative total of particles having a particle size of less thanor equal to about 160 μm, 100 μm, 40 μm, 20 μm, 10 μm, 5 μm, 3 μm, or 2μm. For example, the pharmaceutical composition can have at least about50% of the cumulative total of particles having a particle size of lessthan or equal to about 160 μm, 100 μm, 40 μm, 20 μm, 10 μm, 5 μm, 3 μm,2 μm, 1 μm, or 0.5 μm. For example, the pharmaceutical composition canhave at least about 10% of the cumulative total of the particles havinga particle size of less than or equal to about 160 μm, 100 μm, 40 μm, 20μm, 5 μm, 2 μm, 1 μm, 0.5 μm, or 0.1 μm. In the pharmaceuticalcomposition, the particles can have a median diameter of, for example,less than or equal to about 160 μm, 40 μm, 20 μm, 10 μm, 5 μm, 4 μm 3μm, 2 μm, 1 μm, 0.5 μm, 0.3 μm, or 0.2 μm. For example, the particlescan have a median diameter of from about 0.2 μm to about 50 μm, or amedian diameter of from about 0.5 μm to about 30 μm. For example, thepharmaceutical composition can have the cumulative total of particleshaving a ratio of mean diameter over median diameter of at most about 2μm. The pharmaceutical invention can have particles that include thecompound in a crystalline state, in at least two different polymorphstates.

In some embodiments, the pharmaceutical composition includes a compoundof Formula I or a polymorph thereof in particle form, where the particleor particles are less than 20 micron, 10 micron, 5 micron, 2 micron, 1micron or 0.5 micron.

The present invention provides a substantially pure compound of FormulaII,

wherein each R₁ is independently H, Cl, or F; and n is 0, 1, 2, 3, or 4.In some embodiments, the compound of Formula II is in particle form.

In some embodiments, the substantially pure compound is Compound 1. Insome embodiments, Compound 1 is in particle form.

In some embodiments, the substantially pure compound is selected fromthe group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof.

In some embodiments, the substantially pure compound is a polymorph ofCompound 1. For example, in some embodiments, the polymorph is apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 1. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

In some embodiments, the polymorph of Compound 1 is in particle form.

In some embodiments, the compound, product and or pharmaceuticalcomposition has a purity of at least about 80%, about 85%, about 90%,about 95%, or about 99%. In some embodiments, the compound, product andor pharmaceutical composition has a purity of at least about 95.5%,about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%,about 99%, or about 99.5%. In some embodiments, the compound, productand or pharmaceutical composition has a purity of at least about 99.1%,about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about99.7%, about 99.8%, or about 99.9%.

In some embodiments, the compound, product and or pharmaceuticalcomposition has impurities of at most about 10%, about 5%, about 1%,about 0.15%, or about 0.5%. In some embodiments, the compound, productand or pharmaceutical composition contains, for each single impurity, atmost about 0.5%, about 0.2%, about 0.15%, or about 0.1%. In a furtherembodiment, the impurities are one or more from the group consisting of2-acetyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione,2,6-Diacetyl-naphtho[2,3-b]furan-4,9-dione,2,7-Diacetyl-naphtho[2,3-b]furan-4,9-dione,3-Acetyl-naphtho[2,3-b]furan-4,9-dione, Naphtho[2,3-b]furan-4,9-dione,Naphtho[2,3-b]furan-4,9-dione, Naphtho[2,3-b]furan-4,9-diol, and1-(4,9-Dihydroxy-naphtho[2,3-b]furan-2-yl)-ethanone.

In some embodiments, the impurities include a residual solvent. In someembodiments, the solvent is selected from the group consisting of ethylacetate (EtOAc), toluene, Ethanol, methanol, chloroform, andCH₂Cl₂/hexane.

In some embodiments, the purity is determined with HPLC (HighPerformance Liquid Chromatography). In some embodiments, the purity isdetermined with NMR (Nuclear Magnetic Resonance). In a furtherembodiment, the purity is determined with both HPLC and NMR.

The invention also provides a polymorph of Compound 1 in a particleform, where the compound is in a highly purified form, product and/orpharmaceutical composition. For example, in some embodiments, thepolymorph is a polymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dionecharacterized by an X-ray diffraction pattern substantially similar tothat set forth in FIG. 1. In some embodiments, the polymorph is apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 2. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

The polymorph of Compound 1 is in a particle form. In some embodiments,the polymorph of Compound 1 is in a particle form, where the particlehas a diameter of less than or equal to about 160 μm, about 150 μm,about 120 μm, about 100 μm, about 50 μm, about 40 μm, or about 20 μm. Insome embodiments, the polymorph of Compound 1 in particle form is in apopulation of particles, where the population of particles have a D₅₀(i.e., the median point of the particle size distribution that dividesthe distribution in two equal parts) of less than or equal to about 160μm, about 150 μm, about 120 μm, about 100 μm, about 50 μm, about 40 μm,or about 20 μm. In some embodiments, the polymorph of Compound 1 is in aparticle form, where the particle has a diameter of less than or equalto about 10 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, about 1μm, about 0.5 μm, about 0.2 μm, or about 0.1 μm. In some embodiments,the polymorph of Compound 1 in particle form is in a population ofparticles, where the population of particles have a D₅₀ of less than orequal to about 10 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm,about 1 μm, about 0.5 μm, or about 0.2 μm.

The present invention provides a particle or a population of particlesof a polymorph of Compound 1, which are active, i.e., have an efficacyand/or an antitumor activity. The active particle or particles havecertain size, for example, has a diameter or D₅₀ of less than or equalto about 200 μm, about 150 μm, about 100 μm, about 40 μm, or about 20μm, about 10 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, about 1μm, about 0.5 μm, or about 0.2 μm. The particle or particles that arelarger than the certain size are either inactive or less active than theparticles described herein.

A fraction of the cumulative total of the particles of a polymorph ofCompound 1 can have a diameter or D₅₀ of less than or equal to about 200μm. In some embodiments, a fraction of a set of particles can be atleast about 1%, at least about 5%, at least about 10%, at least about20%, or at least about 30% of the total number of particles in the set.In some embodiments, the fraction is a substantial fraction. Forexample, a “substantial fraction” of a set of particles can be at leastabout 99%, at least about 95%, at least about 90%, at least about 85%,at least about 80%, at least about 75%, at least about 70%, at leastabout 60%, or at least about 50% of the total number of particles in theset.

In some embodiments, the population of particles of a polymorph ofCompound 1 can have at least about 90% of the cumulative total ofparticles having a particle size of less than or equal to about 160 μm,100 μm, 40 μm, 20 μm, 10 μm, 5 μm, 3 μm, or 2 μm, 1 μm or 0.5 μm. Forexample, the population of particles of a polymorph of Compound 1 canhave at least about 50% of the cumulative total of particles having aparticle size of less than or equal to about 160 μm, 100 μm, 40 μm, 20μm, 10 μm, 5 μm, 3 μm, 2 μm, 1 μm, or 0.5 μm. For example, thepopulation of particles of a polymorph of Compound 1 can have at leastabout 10% of the cumulative total of the particles having a particlesize of less than or equal to about 160 μm, 100 μm, 40 μm, 20 μm, 5 μm,2 μm, 1 μm, 0.5 μm, or 0.1 μm. In the population of particles of apolymorph of Compound 1, the particles can have a median diameter of,for example, less than or equal to about 160 μm, 40 μm, 20 μm, 10 μm, 5μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.5 μm or 0.2 μm. For example, the particlescan have a median diameter of from about 0.002 μm to about 50 μm, or amedian diameter of from about 0.2 μm to about 30 μm. For example, thepopulation of particles of a polymorph of Compound 1 can have thecumulative total of particles having a ratio of mean diameter overmedian diameter of at most about 2. The population of particles of apolymorph of Compound 1 can have particles that include the compound ina crystalline state, in at least two different polymorph states.

In some embodiments, the polymorph of Compound 1 is in a particle form,where the particle has a diameter of less than or equal to about 20micron, 10 micron, 5 micron, or 2 3 micron, 2 micron, 1 micron, 0.5micron, 0.2 micron, or 0.1 micron. In some embodiments, the polymorph ofCompound 1 in particle form is in a population of particles, where thepopulation of particles have a D₅₀ of less than or equal to about 20micron, 10 micron, 5 micron, 4 micron, 5 micron, 3 micron, 2 micron, 1micron, 0.5 micron or 0.2 micron.

The present invention also provides a pharmaceutical composition, whichincludes a therapeutically effective amount of the substantially purenaphthofuran compound and a pharmaceutically acceptable carrier,excipient, or diluent. The excipient can include, for example, aglycerol ester of a fatty acid, a glycerol ester of a saturated fattyacid, a glycerol ester of a saturated fatty acid having from 8 to 18carbons, glyceryl laurate, polyethylene glycol, cellulose,microcrystalline cellulose, carboxymethylcellulose, aphosphatidylcholine, a lipid, a sterol, cholesterol, a surfactant, apolysorbate, and/or a polyoxyethylene sorbitan alkylate.

In some embodiments according to the invention, an item of manufacturecan include a container containing a therapeutically effective amount ofthe pharmaceutical composition and a pharmaceutically acceptableexcipient.

A method for producing a compound, product and/or pharmaceuticalcomposition according to some embodiments of the invention can includemilling the compound to form the particles. For example, the compoundcan be ball milled, roll milled, jet milled, wet milled, ultrasonicallymilled, ground, or treated with a combination of these and/or othermilling procedures. The temperature of the compound can be reduced, forexample, reduced to a cryogenic temperature, and milled Such reductionin temperature can render the compound more brittle and more amenable toparticle size reduction by milling.

A method for producing a compound, product and/or pharmaceuticalcomposition according to some embodiments of the invention can includecrystallization. The particle size distribution (PSD) obtained duringcrystallization is influenced by a combination of various mechanismsthat occur during crystallization, such as nucleation, growth,aggregation, attrition, breakage, etc. When the particle size cannot beconsistently controlled during crystallization to meet the desiredspecifications, an extra processing step such as dry milling can beincluded.

A method according to the invention of treating, delaying theprogression of, preventing a relapse of, alleviating a symptom of, orotherwise ameliorating a human, mammal, or animal subject afflicted witha neoplasm can include administering a therapeutically effective amountof the compound, product and/or pharmaceutical composition, so thatanti-neoplastic activity occurs. For example, the anti-neoplasticactivity can be anticancer activity. For example, the anti-neoplasticactivity can include slowing the volume growth of the neoplasm, stoppingthe volume growth of the neoplasm, or decreasing the volume of theneoplasm. The neoplasm can include a solid tumor, a malignancy, ametastatic cell, a cancer stem cell. The neoplasm can include acarcinoma, a sarcoma, an adenocarcinoma, a lymphoma, or a hematologicalmalignancy. The neoplasm can be refractory to treatment by chemotherapy,radiotherapy, and/or hormone therapy. The compound, product and/orpharmaceutical composition can be administered to prevent relapse of theneoplasm. The compound, product and/or pharmaceutical composition can beadministered as an adjuvant therapy to surgical resection. The compound,product and/or pharmaceutical composition can be administered, forexample, orally and/or intravenously.

A method according to the invention also includes treating, delaying theprogression of, preventing a relapse of, alleviating a symptom of, orotherwise ameliorating a disease or disorder in a human, mammal, oranimal subject afflicted with that disease or disorder. In someembodiments, the disease or disorder is selected from the groupconsisting of an autoimmune disease, an inflammatory disease,inflammatory bowel diseases, arthritis, autoimmune demyelinationdisorder, Alzheimer's disease, stroke, ischemia reperfusion injury andmultiple sclerosis.

Administration of the compounds, products and/or pharmaceuticalcompositions to a patient suffering from a disease or disorder isconsidered successful if any of a variety of laboratory or clinicalresults is achieved. For example, administration is consideredsuccessful one or more of the symptoms associated with the disease ordisorder is alleviated, reduced, inhibited or does not progress to afurther, i.e., worse, state. Administration is considered successful ifthe disorder, e.g., an autoimmune disorder, enters remission or does notprogress to a further, i.e., worse, state.

In some embodiments, the compounds, products and/or pharmaceuticalcompositions described herein are administered in combination with anyof a variety of known therapeutics, including for example,chemotherapeutic and other anti-neoplastic agents, anti-inflammatorycompounds and/or immunosuppressive compounds. In some embodiments, thecompounds, products and/or pharmaceutical compositions described hereinare useful in conjunction with any of a variety of known treatmentsincluding, by way of non-limiting example, surgical treatments andmethods, radiation therapy, chemotherapy and/or hormone or otherendocrine-related treatment.

These “co-therapies” can be administered sequentially or concurrently.The compounds, products and/or pharmaceutical compositions describedherein and the second therapy can be administered to a subject,preferably a human subject, in the same pharmaceutical composition.Alternatively, the compounds, products and/or pharmaceuticalcompositions described herein and the second therapy can be administeredconcurrently, separately or sequentially to a subject in separatepharmaceutical compositions. The compounds, products and/orpharmaceutical compositions described herein and the second therapy maybe administered to a subject by the same or different routes ofadministration. In some embodiments, the co-therapies of the inventioncomprise an effective amount of the compounds, products and/orpharmaceutical compositions described herein and an effective amount ofat least one other therapy (e.g., prophylactic or therapeutic agent)which has a different mechanism of action than the compounds, productsand/or pharmaceutical compositions described herein. In someembodiments, the co-therapies of the present invention improve theprophylactic or therapeutic effect of the compounds, products and/orpharmaceutical compositions described herein and of the second therapyby functioning together to have an additive or synergistic effect. Incertain embodiments, the co-therapies of the present invention reducethe side effects associated with the second therapy (e.g., prophylacticor therapeutic agents).

In some embodiments, the disease or disorder can be treated byadministering the compound, product and/or pharmaceutical composition asfollows. The blood molar concentration of the compound can be at leastan effective concentration and less than a harmful concentration for afirst continuous time period that is at least as long as an effectivetime period and shorter than a harmful time period. The blood molarconcentration can be less than the effective concentration after thefirst continuous time period. For example, the effective concentrationcan be about 0.1 μM, about 0.2 μM, about 0.5 μM, about 1 μM, about 2 μM,about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 10 μM, or anotherconcentration determined to be effective by one of skill in the art. Forexample, the harmful concentration can be about 1 μM, about 3 μM, about10 μM, about 15 μM, about 30 μM, about 100 μM, or another concentrationdetermined to be harmful by one of skill in the art. For example, theeffective time period can be about 1 hour, 2 hour, about 4 hours, about6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours,or another time period determined to be effective by one of skill in theart. For example, the harmful time period can be about 12 hours, about24 hours, about 48 hours, about 72 hours, about 144 hours, or anothertime period determined to be harmful by one of skill in the art.

In some embodiments, the therapeutically effective amount of thecompound, product and/or pharmaceutical composition is selected toproduce a blood concentration greater than the IC₅₀ of cells of thetumor and less than the IC₅₀ of normal cells. In some embodiments, thetherapeutically effective amount is selected to produce a bloodconcentration sufficiently high to kill cells of the tumor and less thanthe IC₅₀ of normal cells.

In some embodiments, the compound, product and/or pharmaceuticalcomposition is administered orally in a dosage form, for example, atablet, pill, capsule (hard or soft), caplet, powder, granule,suspension, solution, gel, cachet, troche, lozenge, syrup, elixir,emulsion, oil-in-water emulsion, water-in-oil emulsion, and/or adraught.

In some embodiments according to the present invention, a compositionfor reducing or inhibiting the replication or spread of neoplastic cellsincludes a set of particles selected by the following method. A compoundaccording to Formula I or a salt or solvate thereof can be provided.

In some embodiments, Compound 1 or a salt or solvate thereof can beprovided. In some embodiments, a polymorph of Compound 1 can beprovided. For example, in some embodiments, the polymorph is a polymorphof 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by anX-ray diffraction pattern substantially similar to that set forth inFIG. 1. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

At least one set of particles including the compound can be prepared.The particle size distribution of each at least one set of particles canbe determined. At least one set of particles can be administered toneoplastic cells and to normal cells at a predetermined concentrationand for a predetermined period of time. The effect of the particles onthe metabolism and/or division of the neoplastic cells and the normalcells can be observed. An effectivity rating can be assigned to each setof particles based on the effect of the particles on the neoplasticcells. A toxicity rating can be assigned to each set of particles basedon the effect of the particles on the normal cells. The effectivityrating and/or the toxicity rating of the at least one set of particleshaving a first particle size distribution can be compared with theeffectivity rating and/or the toxicity rating of at least one other setof particles having a particle size distribution different than thefirst particle size distribution. The set of particles having aneffectivity rating greater than, a toxicity rating less than, and/or aweighted effectivity rating and toxicity rating sum greater than the atleast one other set of particles can be selected as an optimum set. Forexample, the particle size distribution of the optimum set of particlescan be identified as an optimum particle size distribution. For example,the optimum set of particles can be included in the composition. Forexample, the effectivity rating can be proportional to antitumoractivity. For example, the effectivity rating can be based on inhibitionof metabolism and/or division of the neoplastic cells. For example, thetoxicity rating can be inversely proportional to tolerability. Forexample, the toxicity rating can be based on inhibition of metabolismand/or division of normal cells. For example, the at least one set ofparticles can be administered to the neoplastic cells and to the normalcells in vitro. For example, the effectivity rating can be the IC₅₀ ofthe neoplastic cells. For example, the toxicity rating can be the IC₅₀of the normal cells. For example, the at least one set of particles canbe administered to the neoplastic cells and to the normal cells in vivoin a test animal. The test animal can be, for example, a mammal,primate, mouse, rat, guinea pig, rabbit, or dog. The effectivity ratingcan be the decrease in volume of the neoplastic cells, and the toxicityrating can be the decrease in mass of the test animal.

In some embodiments, preparing the one set of particles including thecompound can include isolating particles of a predetermined particlesize distribution by dissolving and dispersing the compound, dissolvingand dispersing the compound with a microfluidic technique, dissolvingand dispersing the compound with cavitation or nebulization, milling thecompound, ball milling the compound, roll milling the compound, jetmilling the compound, wet milling the compound, ultrasonically millingthe compound, grinding the compound, and/or sieving the compound. Theparticles can be suspended in a pharmaceutically acceptable excipient.Determining the particle size distribution can include using a techniqueselected from the group consisting of sieve analysis, opticalmicroscopic counting, electron micrograph counting, electroresistancecounting, sedimentation time, laser diffraction, acoustic spectroscopy,and combinations.

A method of treating a neoplasm or other cell proliferation disorder caninclude administering to a human, mammal, or animal afflicted with aneoplasm a therapeutically effective amount of a composition includingan optimum set of particles of the composition having an optimumparticle size and distribution.

The present invention also provides a process of preparing a compound ofFormula II,

wherein R₁ is H, Cl, or F, the process including, reacting a compound ofFormula III,

with a ketone in a first solvent while in the presence of a base,crystallizing crude product from the aged reaction mixture, and,reacting the crude product with an oxidizing agent in a second solvent.

In some embodiments, the reaction is carried out in an open aircontainer.

In some embodiments, the ketone is a compound of Formula IV.

In some embodiments, the first solvent is selected from the groupconsisting of tetrahydrofuran (THF), dioxane, and toluene, and the baseis selected from the group consisting of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethyl amine, anddiisopropylethyl amine. In some embodiments, the oxidizing agent ismanganese dioxide. In some embodiments, the second solvent is toluene.In some embodiments, the process further includes treating the productof oxidization with charcoal.

The present invention provides a process of preparing a compound ofFormula II,

wherein R₁ is H, Cl, or F, the process including, reacting a compound ofFormula III,

with a ketone in a first solvent while in the presence of a base,crystallizing crude product from the aged reaction mixture, dissolvingthe crude product in a second solvent, and, treating the crude productwith charcoal.

The present invention provides a process of preparing a naphthofurancompound. The process includes reacting a naphthodihydrofurane compoundor a mixture including the naphthodihydrofurane compound with anoxidizing agent in a first solvent. In some embodiments, the mixturefurther includes a naphthofuran compound. In some embodiments, thenaphthofuran compound is selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof. In some embodiments, the oxidizing agent is manganesedioxide. In some embodiments, the first solvent is toluene. In someembodiments, the process further includes filtering the oxidizationproduct through a pad of activated carbon. In some embodiments, theprocess further includes crystallizing the naphthofuran compound byevaporating the first solvent. In some embodiments, the process furtherincludes re-crystallizing the naphthofuran compound with a secondsolvent. In some embodiments, the second solvent is ethyl acetate. Insome embodiments, the process further includes slurrying thenaphthofuran compound with a second solvent, heating the slurry, andcooling the slurry.

The present invention provides a process of preparing a substantiallypure naphthofuran compound. The process includes crystallizing anaphthofuran compound with a first solvent, and re-crystallizing thenaphthofuran compound with a second solvent. The present inventionprovides another process of preparing a substantially pure naphthofurancompound. The process includes crystallizing a naphthofuran compoundwith a first solvent, slurrying the crystalline naphthofuran compoundwith a second solvent, heating the slurry, and cooling the slurry. Insome embodiments, the naphthofuran compound selected from the groupconsisting of 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof. In some embodiments, the first solvent is toluene. Insome embodiments, the second solvent is ethyl acetate.

The present invention provides a naphthofuran compound prepared by anyone of the above processes. In some embodiments, the naphthofurancompound is selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof. In some embodiments, the naphthofuran compound has apurity of at least about 80%, about 85% or about 90%, about 95%, orabout 99%. In some embodiments, the naphthofuran compound has impuritiesof at most about 10%, about 5%, about 2%, or about 1%, about 0.5%, about0.2%, about 0.15%, or about 0.1%.

The invention provides methods for preparing particles of Compound 1,including particles of a polymorph of Compound 1, particles of highlypure forms of Compound 1 and particles of highly pure forms of apolymorph of Compound 1. In some embodiments, particles having a desiredmedian particle size, for example, about 20 microns, are produced bymilling crystals of Compound 1, including crystals of a purified form ofCompound 1, crystals of a polymorph of Compound 1 and/or crystals of apurified form of a polymorph of Compound 1. For example, the crystalsare milled using a jet milling method where the venturi pressure isabout 40, the mill pressure is about 100, and the feed rate isapproximately 1304 g/hour.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration depicting XRPD Data of Crystal Form 1.

FIG. 2 is an illustration depicting XRPD Data of Crystal Form 2.

FIG. 3 is an illustration depicting XRPD Data of Crystal Form 3.

FIG. 4 is an illustration depicting the comparison of XRPD Data ofCrystal Form 1 and Crystal Form 3.

FIGS. 5A and 5B are a series of illustrations depicting the syntheticprocess for Crystal Form 2.

FIGS. 6A-6D are a series of illustrations depicting the syntheticprocess for Crystal Form 3.

FIGS. 7A and 7B are photographs depicting the morphology of CrystalForms 1 and 3.

FIG. 8 is a graph depicting the limited anti-tumor activity of CrystalForm 1.

FIG. 9 is a graph depicting the antitumor activity of Crystal Form 2.

FIG. 10 is a graph depicting the comparison of antitumor activity ofCrystal Form 1 and Crystal Form 3.

FIG. 11 is a graph depicting clinical pharmacokinetic (PK) data incancer patients for Crystal Form 2.

FIG. 12 is a graph depicting clinical PK data of Crystal Form 3 incancer patients.

FIG. 13 is a graph depicting the toxicity observed with about 90% pureCrystal Form 2 produced using the synthetic process illustrated in FIGS.5A-5B.

FIG. 14 is a graph depicting the safety of about 95% pure Crystal Form 2produced using the synthetic process illustrated in FIGS. 5A-5B.

FIG. 15 is a graph depicting the anti-tumor activity of Compound 1 withdifferent particle size ranges.

FIG. 16 is a graph depicting in vivo PK data of Compound 1 withdifferent particle size ranges.

FIG. 17 is a graph depicting the relationship between dissolution andparticle size of Compound 1.

FIG. 18 is an illustration depicting the differences betweencancer-stem-cell-specific and conventional cancer therapies.

FIG. 19 is an illustration depicting a complete regression of a coloncancer metastatic lesion to kidney.

FIG. 20 is a graph that illustrates the pharmacokinetics of BID dosingin patients, where the patients were dosed at 500 mg twice daily (1000mg total daily dose).

FIG. 21 is a graph that illustrates the pharmacokinetics of once dailydosing in patients, where the patients were dosed at 20 mg once daily.

FIG. 22 is a graph that illustrates the comparison of progression freesurvival of colorectal cancer patients treated with Compound 1. Theprogression free survival (PFS) of evaluable patients with colorectalcancer treated with Compound 1 was compared against historical PFS datafor best supportive care in patients with colorectal cancer.

FIG. 23 is a graph that illustrates the comparison of progression freesurvival (PFS) versus pharmacokinetic exposure. The PFS of evaluablepatients receiving Compound 1 was compared against Compound 1 exposureabove or below 1.6 uM for at least 4 hours.

FIG. 24 is a graph that illustrates the desirable PK pattern forimproved safety and efficacy.

FIG. 25 is a photograph illustrating that patients achieved prolongstable disease (>16 weeks) during BBI608 treatment have high levels ofp-STAT3 in their tumor tissues prior to the treatment.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. A person skilled in the relevant artwill recognize that other equivalent components can be employed andother methods developed without parting from the spirit and scope of theinvention. All references cited herein are incorporated by reference asif each had been individually incorporated.

In this text, a “substantial fraction” of a set of particles can be atleast about 99%, at least about 95%, at least about 90%, at least about85%, at least about 80%, at least about 75%, at least about 70%, atleast about 60%, or at least about 50% of the total number of particlesin the set.

The anti-cancer stem cell activity of a composition can be determined invitro or in vivo. For example, antitumor activity of a composition canbe determined in vitro by administering the compound and measuring theself-renewal and survival of cancer stem cells, For example, theantitumor activity of a compound can be assessed in vitro by comparingthe behavior of tumor cells to which the compound has been administeredwith the behavior of tumor cells to which the compound has not beenadministered (a control). For example, antitumor activity of acomposition can be determined in vivo by measuring, in an animal towhich the compound has been administered, the change in volume of atumor, by applying a metastatic model, and/or by applying an orthotopicmodel. For example, the antitumor activity of a compound can be assessedin vivo by comparing an animal to which the compound has beenadministered to an animal to which the compound has not beenadministered (a control).

The tolerability of a composition can be determined in vitro or in vivo.For example, tolerability of a composition can be determined in vitro byadministering the compound and measuring the division rate of normalcells, by measuring the nutrient uptake of normal cells, by measuringindicators of metabolic rate of normal cells other than nutrient uptake,by measuring the growth of normal cells, and/or by measuring anotherindicator of the vitality of normal cells. For example, the tolerabilityof a compound can be assessed in vitro by comparing the behavior ofnormal cells to which the compound has been administered with thebehavior of normal cells to which the compound has not been administered(a control). For example, tolerability of a composition can bedetermined in vivo by measuring, in an animal to which the compound hasbeen administered, body weight or food intake or making clinicalobservations, such as hair retention or loss, activity, and/orresponsiveness to stimuli. For example, the tolerability of a compoundcan be assessed in vivo by comparing an animal to which the compound hasbeen administered to an animal to which the compound has not beenadministered (a control).

A compound, product and/or pharmaceutical composition can be assigned aneffectivity rating and/or a toxicity rating. For example, theeffectivity rating can be proportional to antitumor activity or can be amonotonically increasing function with respect to antitumor activity.For example, the toxicity rating can be inversely proportional totolerability or can be a monotonically decreasing function with respectto tolerability. A naphthofuran compound has been reported to lack invivo antitumor activity. See, M. M. Rao and D. G. I. Kingston, J.Natural Products, 45(5) (1982) 600-604. Furthermore, the compound hasbeen reported to be equally toxic to cancer cells and normal cells. Thatis, the compound was reported as killing both cancer cells and normalcells equally, concluding the compound has no potential for cancertreatment. See, K. Hirai K. et al., Cancer Detection and Prevention,23(6) (1999) 539-550; Takano A. et al., Anticancer Research 29:455-464,2009.

However, experimental studies reported herein indicate that when thecompound is administered as particles having an appropriate particlesize distribution to achieve a certain pharmacokinetic exposure asdescribed in this publication, the compound does have selectiveantitumor activity.

For the purposes of the present invention, “bioavailability” of a drugis defined as the relative amount of drug from an administered dosageform which enters the systemic circulation and the rate at which thedrug appears in the blood stream. Bioavailability is governed by atleast three factors: (i) absorption which controls bioavailability,followed by (ii) its tissue re-distribution and (iii) elimination(metabolic degradation plus renal and other mechanisms).

“Absolute bioavailability” is estimated by taking into considerationtissue re-distribution and biotransformation (i.e., elimination) whichcan be estimated in turn via intravenous administration of the drug.Unless otherwise indicated, “HPLC” refers to high performance liquidchromatography; “pharmaceutically acceptable” refers to physiologicallytolerable materials, which do not typically produce an allergic or otheruntoward reaction, such as gastric upset, dizziness and the like, whenadministered to a mammal; “mammal” refers to a class of highervertebrates including man and all other animals that nourish their youngwith milk secreted by mammary glands and have the skin usually more orless covered with hair; and “treating” is intended to encompassrelieving, alleviating, or eliminating at least one symptom of adisease(s) in a mammal.

The term “treatment”, as used herein, is intended to encompassadministration of compounds according to the invention prophylacticallyto prevent or suppress an undesired condition, and therapeutically toeliminate or reduce the extent or symptoms of the condition. Treatmentalso includes preventing the relapse of an undesired condition, delayingthe progression of an undesired condition, and preventing or delayingthe onset of an undesired condition. Treatment according to theinvention is given to a human or other mammal having a disease orcondition creating a need of such treatment. Treatment also includesapplication of the compound to cells or organs in vitro. Treatment maybe by systemic or local administration.

An effective amount is the amount of active ingredient administered in asingle dose or multiple doses necessary to achieve the desiredpharmacological effect. A skilled practitioner can determine andoptimize an effective dose for an individual patient or to treat anindividual condition by routine experimentation and titration well knownto the skilled clinician. The actual dose and schedule may varydepending on whether the compositions are administered in combinationwith other drugs, or depending on inter-individual differences inpharmacokinetics, drug disposition, and metabolism. Similarly, amountsmay vary for in vitro applications. It is within the skill in the art toadjust the dose in accordance with the necessities of a particularsituation without undue experimentation. Where disclosed herein, doseranges do not preclude use of a higher or lower dose of a component, asmight be warranted in a particular application.

The descriptions of pharmaceutical compositions provided herein includepharmaceutical compositions which are suitable for administration tohumans. It will be understood by the skilled artisan, based on thisdisclosure, that such compositions are generally suitable foradministration to any mammal or other animal. Preparation ofcompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modifications with routine experimentation basedon pharmaceutical compositions for administration to humans.

Compound Structure and Properties

A naphthofuran compound of Formula I, such as2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, was practically insoluble inwater and a broad panel of solvents tested, including DMSO (dimethylsulfoxide), N-methylpyrrolidine, DMA (dimethylacetamide), ethanol,PEG400 (polyethylene glycol 400), propylene glycol, Cremophor EL(polyethoxylated castor oil), Labrasol (CaprylocaproylMacrogolglycerides (Polyoxylglycerides)), Labrafil M (vegetable oilPEG-6 (polyethylene glycol) ester), and Capryol (propylene glycolcaprylate). The naphthofuran compound may be soluble in a range of polarorganic solvents, such as certain halocarbons, e.g., chlorocarbons, likemethylene chloride, esters, ethyl acetate, carboxylic acids, like aceticacid, ketones, like acetone, and alcohols, like methanol. Thenaphthofuran compound was found to be soluble in methylene chloride andethyl acetate.

The experimental studies described herein, which found that selectiveantitumor activity was achieved by administering the active compound ofa pharmaceutical composition in the form of small particles to achieve acertain pharmacokinetic exposure for selective anticancer activity,focused on a naphthofuran compound. Given the presently discussedobservations made with the compound, other naphthofurans, for example,naphthofurans, may similarly exhibit an advantageous modification oftheir pharmacokinetic profiles to the achievement of a certainpharmacokinetic exposure to achieve selective anti-cancer activity whenadministered in the form of particles of small diameter. Thepharmacokinetic profile of other naphthofurans administered as one ormore different particle size distributions can be experimentallydetermined.

Some other compounds that may exhibit an improvement in theirpharmacokinetic profile and efficacy with a decrease in particle size ofthe form in which they are administered to an animal, a mammal, or ahuman, as observed for the compound tested in examples, include thosepresented as Formula I, and salts and solvates thereof.

In Formula I, the notation (R₁)_(n) indicates that an (R₁) substituentis independently substituted at each available position along thebenzene ring. For example, with n equal to 4, the four R₁ substituentsmay all be the same, or they may each be different from any other. Forexample, each (R₁) can be independently selected from the groupconsisting of hydrogen, halogen, fluorine, cyano, nitro, CF₃, OCF₃,alkyl, methyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heterocycle, substituted heterocycle, aryl,substituted aryl, OR_(a), SR_(a), and NH₂. Alkyl can include moietieshaving, for example, from 1 to 8 carbon atoms connected by single bonds,alkenyl can include moieties having, for example, from 2 to 8 carbonatoms connected by one or more double bonds, and alkynyl can includemoieties having, for example, from 2 to 8 carbon atoms connected by oneor more triple bonds. Substituents can include moieties such ashydrogen, halogen, cyano, nitro, aryl, OR_(a), SR_(a), and NH₂. Forexample, each (R₁) can be independently selected from the groupconsisting of hydrogen, methyl, F (fluorine), Cl (chlorine), Br(bromine), I (iodine), OH (hydroxyl), and NH₂ (amine). For example, R₃can be selected from the group consisting of hydrogen, halogen,fluorine, cyano, CF₃, OCF₃, alkyl, methyl, substituted alkyl,halogen-substituted alkyl, hydroxyl-substituted alkyl, amine-substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heterocycle, substituted heterocycle, aryl, substitutedaryl, OR_(a), SR_(a), and NR_(b)R_(c). For example, R₃ can be selectedfrom the group consisting of methyl and C(R₈)₃. Each (R₈) can beindependently selected from the group consisting of hydrogen, methyl, F(fluorine), Cl, Br, I, OH, and NH₂. For example, at most two of theindependently selected (R₁) substituents and the (R₈) substituents canbe selected to be F (fluorine), with the remainder being selected to behydrogen.

In some embodiments, the compound of Formula I is selected from thegroup consisting of 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, an enantiomer, diastereomer,tautomer, and a salt or solvate thereof. For example, each (R₁) can beselected to be hydrogen and R₃ can be selected to be methyl, so that thecompound of Formula I is 2-acetylnaphtho[2,3-b]furan-4,9-dione. Forexample, each R_(a) can be independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocycle,substituted heterocycle, aryl, and substituted aryl. For example, eachR_(b) and R_(c) can be independently selected from the group consistingof, hydrogen, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, heterocycle, substituted heterocycle, aryl, and substitutedaryl. Alternatively, an R_(b) and R_(c) together with the N to whichthey are bonded can form a heterocycle or substituted heterocycle.

Polymorphs

Naphthofuran compounds of the invention include polymorphs. In someembodiments, the polymorph is a polymorph of a compound according toFormula I. In some embodiments, the polymorph is a polymorph ofCompound 1. For example, in some embodiments, the polymorph is apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 1. This polymorph is referred to herein as “Crystal Form 1,”“Form 1,” or “XRPD1” and these terms are used interchangeably. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 2.This polymorph is referred to herein as “Crystal Form 2,” “Form 2,” or“XRPD2” and these terms are used interchangeably. In some embodiments,the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3.This polymorph is referred to herein as “Crystal Form 3,” “Form 3,” or“XRPD3” and these terms are used interchangeably.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.4, 11.9, 14.1, 14.5, 17.3, 21.0, 22.2, 24.0, 26.0, and 28.1 degrees2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 10.2,11.9, 14.1, 14.5, 17.3, 22.2, and/or 28.1 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 10.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 11.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 14.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 17.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 22.2 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.1 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 10.2 degrees 2θ, a peak at least at about 11.9 degrees 2θ, a peakat least at about 14.1 degrees 2θ, a peak at least at about 14.5 degrees2θ, a peak at least at about 17.3 degrees 2θ, a peak at least at about22.2 degrees 2θ, and a peak at least at about 28.1 degrees 2θ and anycombinations thereof.

For example, in some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 11.4, 12.3, 15.0, 23.0, 23.3, 24.1, 24.6, 25.0, 26.1, 27.0, and28.4 degrees 2θ. In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including one or more peaks at least at about 7.5,9.9, 12.3, 15, 23.0, 23.3, 24.6 and/or 28.4 degrees 2θ. In someembodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 7.5 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 9.9 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 12.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 15 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23 degrees 2θ. Insome embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 23.3 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 24.6 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including a peak at least at about 28.4 degrees 2θ.In some embodiments, the polymorph is a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern including two or more peaks from a peak at least atabout 7.5 degrees 2θ, a peak at least at about 9.9 degrees 2θ, a peak atleast at about 15 degrees 2θ, a peak at least at about 12.3 degrees 2θ,a peak at least at about 23.0 degrees 2θ, a peak at least at about 23.3degrees 2θ, a peak at least at about 24.6 degrees 2θ and a peak at leastat about 28.4 degrees 2θ and any combinations thereof.

Crystal Form 1 has been detected in a variety of solvents andconditions, but has been shown to have low anti-tumor activity (FIG. 8).In the studies shown in FIG. 8, immunosuppressed mice with establishedsubcutaneous FaDu human head and neck cancer were given indicated amountof hand grounded Compound 1 with Crystal Form 1, or vehicle controlorally (po). Compound 1 was formulated in GELUCIRE™. All regimens wereadministered daily (qd). Tumor sizes were evaluated periodically duringtreatment.

Crystal Form 2 was obtained surprisingly in the presence of an impurity,and this polymorph has been shown to exhibit potent anti-tumor activity(FIG. 9). In the study shown in FIG. 9, immunosuppressed mice withestablished subcutaneous FaDu human head and neck cancer were given 100mg/kg of micronized Compound 1 produced with the synthetic processdescribed in FIGS. 5A and 5B (first crop), or vehicle control orally(po). Compound 1 was formulated in GELUCIRE™. All regimens wereadministered daily (qd). Tumor sizes were evaluated periodically duringtreatment. Form 2 has been successfully manufactured by a current goodmanufacturing practice (cGMP) process and received approval from the FDAand Health Canada to be used in clinical trials. Form 2 has showndesirable pharmacokinetics (FIG. 11), safety, and strong signs ofanti-tumor activity in cancer patients.

Crystal Form 3 has been shown to share a similar, but different, X-raypowder diffraction (XRPD) pattern as Form 1, and displayed verydifferent crystalline habit than Form 1 (FIGS. 7A and B). Form 3 canonly be generated from Form 1 using a specially designed slurry processdescribed herein. Form 3 has been shown to exhibit potent antitumoractivities (FIG. 10). In the study shown in FIG. 10, immunosuppressedmice with established subcutaneous FaDu human head and neck cancer weregiven 200 mg/kg of Compound 1 with hand grounded Crystal Form 1 or Form3, or vehicle control orally (po). Compound 1 was formulated ingelucire. All regimens were administered daily (qd). Tumor sizes wereevaluated periodically during treatment. This polymorph has beensuccessfully manufactured by a cGMP process and received approval fromFDA and Health Canada to be used in clinical trials. Form 3 has alsoshown desirable pharmacokinetics (FIG. 12), safety, and strong signs ofanti-tumor activity in cancer patients.

The synthetic process for preparing Crystal Form 2 is shown in FIGS.5A-5B. Briefly, charged 3-butene-2-one (451.2 grams) is added to a 2liter 3 neck round bottom flask equipped with a mechanical stirrer,thermometer, and addition funnel. To the addition funnel is addedbromine (936.0 grams). After the contents in the flask have cooled to−5° C., the bromine is dropped into the flask with vigorous stirring andmaintaining temperature at −5° C. over 30 minutes. The mixture isstirred for an additional 15 minutes at −5° C., and then is split into 4equal portions. Each portion of the mixture along with tetrahydrofuran(2133.6 grams) is loaded into a 22 liter 4 neck round bottom flaskequipped with a mechanical stirrer, thermometer, and addition funnel.Charged DBU (1,3-Diazabicyclo[5.4.0]undec-7-ene, 222.9 grams) is addedto the addition funnel. The DBU is dropped into the flask with vigorousstiffing and maintaining temperature at 0° C.-5° C. over 30 minutes. Themixture is stirred for an additional 15 min at 0° C.-5° C.2-hydroxy-1,4-naphthoquinone (231 grams) is then added into the flask.Additional DBU (246.0 grams) is charged into the addition funnel andthen dropped into the mixture in the flask at such a rate that thetemperature of the reaction mixture does not exceed 40° C. After theaddition of DBU is complete, the resulting mixture is stirred overnightat room temperature, and a sample of the reaction mixture is taken forHPLC analysis. To the reaction mixture, water (10.8 liters) is charged,and the resulting mixture is cooled to 0° C.-3° C. for at least 30minutes, and then filtered via vacuum filter. The filtered solid isrinsed with 5% aqueous sodium bicarbonate (3 liters), water (3 liters),1% aqueous acetic acid (3 liters) and ethanol twice (2×1 liter)successively. The rinsed solid is stored and pooled together from otherbatches. The combined crude product (28.73 kg) is loaded along withethyl acetate (811.7 kg) into a 500 gallon vessel equipped with amechanical stirrer, thermometer, and a condenser. Under nitrogenatmosphere, the mixture is heated to reflux (72° C.) for 2 hours, andthen filtered with a 10 micron cartridge filter containing an activecarbon layer to remove insolubles. Fresh hot ethyl acetate (10 kg) isused to rinse the vessel, transfer line and filter. The combinedfiltrate is cooled to 0-5° C. and held at this temperature for 2 hours,and then is filtered with 20 inch Buchner filter. The filtered solidproduct is rinsed with 0-5° C. ethyl acetate (5.7 kg), and dried undervacuum at 40° C. to a constant weight. The remaining filtrate is reducedin volume by 63% by evaporation, and the crystallization process isrepeated again to generate a second crop of product which was also driedunder the same condition as the first crop of product. Both cropsobtained are Crystal Form 2. The first crop produced (0.5 kg) had a99.5% purity by HPLC (˜95% by NMR). The second crop produced (1.09 kg)had a 98.9% purity by HPLC (˜90% by NMR).

The synthetic process for preparing Crystal Form 3 is shown in FIGS.6A-6D. The steps are outlined briefly herein. Step 1: 3-Butene-2-one(methyl vinyl ketone, MVK) is brominated using bromine No additionalsolvent is used. The intermediate 3,4-dibromobutan-2-one is dissolved intetrahydrofuran (THF) and reacted with1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to form a second intermediate,3-bromo-3-buten-2-one. Once this reaction is complete,2-hydroxy-1,4-naphthoquinone (HNQ) is added. A second portion of DBU isadded, and the mixture is exposed to air. The reaction is quenched withwater and the solids are collected by filtration. These solids arewashed with aqueous sodium bicarbonate, aqueous acetic acid, water, andethanol. The product is isolated by slurrying in ethanol and collectingthe solids. Step 2: Residual amounts of the2-acetyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione that accompany thedesired 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione (Compound 1) areoxidized to Compound 1 with activated manganese dioxide in toluene. Themixture is filtered through a cake of charcoal and Celite. The filtrateis concentrated to precipitate the product, which is filtered and dried.Step 3: The solids are slurried in ethyl acetate (25 mL/g purifiedCompound 1) at 75° C.-80° C. for about 5 hr, collected by filtration,and dried. Compound 1 produced with this method is Crystal Form 3.Compound 1 produced with this method without the slurry process yieldedCrystal Form 1.

Effect of Compound Particle Size Distribution on Blood Plasma DrugConcentration and Selective Antitumor Activity

Prior to the instant invention, no microparticles of Compound 1 had beencreated and/or evaluated. Previous studies had shown Compound 1 to beequally toxic to normal and cancer cells, and no antitumor activity wasobserved in animal model. The studies presented herein demonstrate thatparticle size reduction of Compound 1 not only improved bioavailability,but also led to increased selective anti-tumor activity without signs oftoxicity. This is unexpected since improvement on bioavailability wouldincrease exposure to Compound 1 equally by cancer cells and normalcells. The mechanism for the selective enhancement of anticanceractivity without enhancement of toxicity to normal cells was not known.In these studies, the improvement in bioavailability of Compound 1appeared to be maximized when the D₅₀ (i.e., the median point of theparticle size distribution that divides the distribution in two equalparts) is about 20 μm. However, further studies were conducted where theD₅₀ value was about 2 μm. Microparticles of Compound 1 having a D₅₀ of 2microns had surprisingly enhanced anti-tumor activity, even though thereis no improvement in pharmacokinetic exposure as compared to particleswith a D₅₀ of 20 microns. In additional studies, nanoparticles ofCompound 1 having a D₅₀ of about 100 nanometers (D50=110.4 nanometers)were created, but surprisingly, a reduction of anti-tumor activity wasobserved with this particle size of Compound 1. Accordingly, in apreferred embodiment, compositions that contain particles of Compound 1,e.g., microparticles, have a D₅₀ equal to or below 20 microns and equalto or above 0.2 microns and possesses surprisingly potent anti-tumoractivity without increase in cytotoxicity to normal cells.

The anti-tumor activity of particles of Compound 1 with differentparticle size ranges is illustrated in FIG. 15, and the pharmacokineticdata for particles of Compound 1 with different particle size ranges isillustrated in FIGS. 16-18. In the study shown in FIG. 15,immunosuppressed mice with established subcutaneous FaDu human head andneck cancer were given indicated amount of Compound 1 with indicatedparticle size, or vehicle control orally (po). All regimens wereadministered daily (qd). Tumor size was evaluated periodically.

Administering the naphthofuran compound in the form of particles havingdefined particle size, e.g., a reduced particle size, was found toenhance plasma drug concentration in vivo. Herein, unless otherwisenoted, the terms “size” and “diameter” will be used interchangeably todescribe particles. It is to be understood that the use of the term“diameter” does not necessarily imply that a particle has a perfectly orapproximately spherical form. For example, “diameter” can be used as anapproximation of the size of a particle, for example, the diameter of asphere of equivalent volume to a non-spherical particle.

In a surprising result, the administration of the naphthofuran compoundparticles of a defined particle size distribution, e.g., as smallparticles, in a pharmaceutical composition was found to result inselective antitumor activity. For example, the compound administered asparticles having a median particle size of 20 μm (i.e., microns, theseterms are used interchangeable herein) showed efficacy (selectiveantitumor activity), although relative weak, in mouse xenograft models.In comparison, the particles of 150 μm (microns) showed no efficacy. Thediscovery that the administration of the naphthofuran compound in theform of smaller particles can result in selective antitumor activity issurprising, and cannot be explained on the basis of an improvement insolubility or bioavailability alone. That is, in general, improvedsolubility is associated with increased drug oral bioavailability, whichcan enhance toxicity to normal cells as well as antitumor activity. Asdiscussed above, the naphthofuran compound can be equally toxic to tumorcells and normal cells if the exposure is not carried out under definedconditions as described in WO 2009/036099 and WO 2009/036101.

In a further surprising result, the administration of the naphthofurancompound particles of a further reduced size, in a pharmaceuticalcomposition was found to result in a significantly improved antitumoractivity but almost an unaltered pharmacokinetic profile, i.e.,unaltered bioavailability. For example, the compound administered asparticles having a median particle size of 2 μm (microns) showeddramatically enhanced efficacy in mouse xenograft models. In comparisonwith the particles of 20 μm, the particles of 2 μm showed significantlyimproved efficacy but very similar pharmacokinetic profile. In otherwords, such an improved efficacy is independent of pharmacokineticprofile, i.e., bioavailability. The result is very surprising, becausefor such a compound with poor solubility, improved efficacy is usuallyassociated with increased drug oral bioavailability.

The observed improvement in the selective antitumor activity istherefore surprising and unexpected. The present invention provides aparticle or particles of a naphthofuran compound, for example, acompound of Formula I, which are active, i.e., have an efficacy or aselective antitumor activity. The active particle or particles have adefined particle size, for example, has a diameter of less than or equalto about 200 μm, about 150 μm, about 100 μm, about 40 μm, or about 20μm, about 10 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, about 1μm, about 0.5 μm, about 0.2 μm, or about 0.1 μm. The particle orparticles that are larger than the defined particle size are eitherinactive or less active than the particles described herein.

Thus, the administration of the naphthofuran compound or anotherCompound according to Formula I in the form of smaller particles canresult in an improvement in its selective antitumor activity. The use ofparticles of a compound according to Formula I having a defined particlesize distribution in dosing can allow for the establishment of desiredselective antitumor activity. For example, the use of the naphthofurancompound particles having a defined particle size distribution, forexample, being smaller particles, can result in a higher bloodconcentration for a shorter period of time, and a selective antitumoractivity, although relative weak. Further reduced particle size of thecompound can lead to significantly improved efficacy with unalteredblood plasma concentration of the compound.

Herein, unless otherwise indicated, the term “blood plasmaconcentration”, “blood molar concentration”, and “blood concentration”are used interchangeably. The term “neoplasm” can be used to describecells which exhibit an abnormal pattern of growth. Such a neoplasm caninclude tumors, both benign and malignant, e.g., solid tumors, as wellas other cell growth disorders, such as leukemia, that have no definedshape and are not confined to a specific region of a human or animalbody. Thus, “neoplasm” includes both cancerous and non-cancerousneoplastic cells and tissues. Herein, unless otherwise stated, madeclear, or referring to a specific study or experiment, the terms “tumor”and “cancer” are to be understood as referring to the broader class ofall neoplasms, including those that are not confined to a specificregion of a human or animal body. However, the more limited term “solidtumor” is to be understood as not including cell growth disorders, suchas leukemia, that have no defined shape and are not confined to aspecific region of a human or animal body.

A neoplasm can exhibit none, one, or more than one of the followingcharacteristics: solid form (a solid tumor), malignancy, metastasis, orStat 3 pathway activity. A neoplasm can, for example, include a cancerstem cell. A neoplasm can be, for example, a carcinoma, sarcoma,adenocarcinoma, lymphoma, or a hematological malignancy.

Absorption has been defined as the process by which a drug is taken fromthe site of administration to the site of measurement within the body.See, M. Rowland, T. N. Tozer (1995) Clinical pharmacokinetics: Conceptsand applications. Lippincott Williams & Wilkins. Oral drug absorption isoften referred to as drug transfer across the apical membrane of theenterocyte, because the apical membrane is considered to be the ratelimiting step for permeation of the membrane. See, U. Fagerholm & H.Lennernäs (1995) Experimental estimation of the effective unstirredwater layer thickness in the human jejunum, and its importance in oraldrug absorption, Eur J Pharm Sci 3: 247-253; M. B. Lande, J. M. Donovan& M. L. Zeidel (1995) The relationship between membrane fluidity andpermeabilities to water, solutes, ammonia, and protons, J Gen Physiol106: 67-84. Permeability is a general term describing how readily thedrug is transferred through a membrane. The specific permeabilitycharacteristics of a drug are dependent on its physico-chemicalproperties, including lipophilicity, charge, size, and polar surfacearea. See, Rowland & Tozer 1995; C. A. Lipinski, F. Lombardo, B. W.Dominy & P. J. Feeney (2001) Experimental and computational approachesto estimate solubility and permeability in drug discovery anddevelopment settings, Adv Drug Deliv Rev 46: 3-26. The rate ofabsorption is dependent on the permeability of the drug, surface area ofthe membrane, and the concentration gradient over the membrane. Theconcentration gradient is the driving force for passive diffusion, themost common mechanism for drug membrane transport. For oraladministration, the drug is mainly absorbed by intestine. Humanintestine is about 5-8 meters long and has a total surface area ofalmost 200 square meters while mouse intestine is only about 10-20 cmlong. Therefore, one can predict that a drug with a larger particle sizemay have a higher or same absorption rate in human as a drug with asmaller particle size does in mouse, despite the permeability of thedrug with a larger particle size being lower than that of the drug witha smaller particle size.

For example, a distribution of particle sizes of a compound according toFormula I, having a median diameter of less than or equal to about 200μm, 150 μm, 100 μm, 80 μm, 60 μm, 40 μm, 20 μm, 10 μm, 5 μm, 4 μm, 3 μm,2 μm, 1 μm, 0.5 μm or 0.2 μm can be predicted to result in a selectiveantitumor activity when administered in a pharmaceutical formulation,e.g., for the treatment of a cancer or tumor. For example, thedistribution of particle sizes can be such that the particles have amedian diameter of from about 0.02 μm to about 5 μm, or from about 0.2μm to about 4 μm. For example, the distribution of particle sizes can besuch that the particles have a median diameter of less than or equal toabout 5 μm, a ratio of mean diameter over median diameter of at mostabout 2, and a ratio of mode diameter over median diameter of at leastabout 0.25.

The term “particle” can refer to an aggregate of a compound of FormulaI. The term “mean” can refer to the sum of the sizes of all particlesdivided by the total number of particles. The term “median” can referto, e.g., a diameter of which one-half of the particles have a greaterdiameter and one-half of the particles have a lesser diameter. The term“mode” can indicate the most frequently-occurring particle size value.The term “cumulative total” can refer to all particles.

The selective antitumor activity achieved by administration of thenaphthofuran compound particles may depend not only on the sizedistribution of the particles, e.g., the volumes of particles ordiameters representative of those volumes, but also on the shape anddistribution of shapes of the particles. For example, a set of particleshaving a needle-like shape may result in a different pharmacokineticprofile than a set of particles having a spherical shape. Thus, it maybe desirable to measure the shape and shape distribution of theparticles to be administered and/or use a process that producesparticles with predetermined shape and shape distribution, for example,a nearly uniform shape, e.g., the particles being approximations ofspheres. For example, the sphericity, ψ, of a particle can be defined as

${\Psi = \frac{{\pi^{1/3}\left( {6\; V_{P}} \right)}^{2/3}}{A_{P}}},$

where V_(p) is the volume of the particle and A_(p) is the surface areaof the particle. A sphere has a sphericity of ψ=1, and the closer thesphericity of a particle is to unity, the more closely the shape of theparticle approximates a sphere. By way of comparison, a tetrahedron hasa sphericity of about 0.671, a cube has a sphericity of about 0.806, anoctahedron has a sphericity of about 0.846, a dodecahedron has asphericity of about 0.910, and an icosahedron has a sphericity of about0.939. Because the form of a sphere minimizes surface area for a givenvolume, a particle that is nearly spherical may be expected to dissolvemore slowly than a particle of the same volume that is less nearlyspherical. The mean sphericity of a set of spheres can be defined as

${\Psi_{m} = \frac{{\pi^{1/3}\left( {6{\sum V_{P}}} \right)}^{2/3}}{\sum A_{P}}},$

where ΣV_(p) is the total volume of all the particles and ΣA_(p) is thetotal surface area of all the particles. For example, particles of acompound according to Formula I administered may have a mean sphericityof at least about 0.8, or a mean sphericity of at least about 0.9.

The size, size distribution, shape, shape distribution, and factors suchas surface roughness or irregularity of the particles can affect themean specific surface area of the set of Compound 1 particlesadministered in a pharmaceutical formulation. The mean specific surfacearea can be defined as ΣA_(p)/Σm_(p), where ΣA_(p) is the total surfacearea of the particles and Σm_(p) is the total mass of the particles. Thegreater the mean specific surface area of the particles, the faster theexpected dissolution of the particles.

The particles of a compound according to Formula I in a pharmaceuticalformulation can include the naphthofuran compound in a crystalline stateacross different particles or within the same particle. The crystallinestate may include one or more polymorphs, across different particles orwithin the same particle. As will be appreciated by one of skill in theart, it is expected that the dissolution rate of the particles can beeffected by the state of matter in the compound particles, for example,whether crystalline, of a first polymorph, or a second polymorph.

One or more of a range of techniques can be applied to determine thesize and/or size distribution of particles of a compound according toFormula I in a pharmaceutical formulation. For example, sieve analysis,optical microscopic counting, electron micrograph counting,electroresistance counting, sedimentation time, laser diffraction,and/or acoustic spectroscopy can be applied. Some or all of thesetechniques or variations thereof can be applied to determine the shape,shape distribution, and/or specific area of the naphthofuran compoundparticles in a pharmaceutical formulation. A BET isotherm and/or airpermeability specific surface technique can be applied to determine thespecific area of particles of a compound according to Formula I in apharmaceutical formulation.

Processes for Generating Naphthofuran Compounds

WO 2009/036099 and WO 2009/036101 disclose a process for the preparationof a naphthofuran compound of Formula II as follows.

In this process, 3-bromo-3-buten-2-one (4-3) is reacted with2-hydroxy-1,4-naphthoquinone (4-4) in an open air container, resultingin 2,3-dihydronaphtho[2,3-b]furan-4,9-dione (4-5).2,3-dihydronaphtho[2,3-b]furan-4,9-dione (4-5) is oxidized by oxygenfrom open air to become naphtho[2,3-b]furan-4,9-dione (4-6). Withnaphtho[2,3-b]furan-4,9-dione produced by this process. However, duringfurther development of the compound, it was determined that this processstill generated significant various impurities which hinders thepotential clinical applications of these compounds. In some embodiments,one of the impurities is 2,3-dihydronaphtho[2,3-b]furan-4,9-dione (4-5).

In one aspect, the present invention provides an improved process forthe preparation of naphthofuran. The improved process minimizes theimpurities, and thereby produces substantially pure naphthofuran. Asused herein the term “substantially pure” refers to a preparationincluding at least about 80% or more, measured as % area HPLC, of thecompound of the present invention. In some embodiments, the naphthofuranis naphtho[2,3-b]furan-4,9-dione and its related compounds (4-6).

In some embodiments, the process of the present invention includesoxidizing the crude product of coupling of 3-bromo-3-buten-2-one (4-3)and 2-hydroxy-1,4-naphthoquinone (4-4) with an oxidizing agent in afirst solvent. In a further embodiment, the oxidizing agent is manganesedioxide (MnO₂). In an even further embodiment, the crude product isisolated before it is oxidized. In some embodiments, the first solventis toluene or chloroform.

In some embodiments, the process of the present invention furtherincludes treating the aged oxidation mixture with charcoal to get rid ofcertain impurities. In a further embodiment, the aged oxidation mixtureis filtered with a pad of activated carbon. In an even furtherembodiment, the mixture is filtered at around 100° C.

In some embodiments, the process of the present invention furtherincludes crystallizing the product from the filtrate. In a furtherembodiment, the product is crystallized by concentrating the filtratewith evaporation, and cooling down.

In some embodiments, the process of the present invention furtherincludes re-crystallizing the product with a second solvent. In afurther embodiment, the second solvent is ethyl acetate.

In an alternative embodiment, the process of the present inventionfurther includes slurrying in a second solvent the product crystallizedfrom the first solvent, heating the slurry, and cooling the slurry. In afurther embodiment, the second solvent is ethyl acetate. In someembodiments, the product is slurried and heated only to partialdissolution. In a further embodiment, the volume of the second solventused to slurry the product is about 1/10, ⅕, ¼, ⅓, ½, or ⅔ of the volumefor the complete dissolution of the product in the heated condition.

The present invention also provides a naphthofuran compound prepared bythe process of the present invention. In some embodiments, thenaphthofuran compound is selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof. In a further embodiment, the naphthofuran compound isprepared by the process including reacting the isolated crude product ofthe coupling of 2-hydroxy-1,4-naphthoquinone (4-4) and3-Bromo-3-buten-2-one (4-3) with manganese dioxide in the presence oftoluene. In an even further embodiment, the process further includesfiltering the aged reaction mixture with a pad of activated carbon.

In another aspect, the present invention provides substantially purenaphthofuran compounds.

In some embodiments, the present invention provides a substantially purecompound selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof.

In some embodiments, the present invention provides a substantially purecompound of Formula II,

wherein each R₁ is independently H, Cl, or F; and n is 0, 1, 2, 3, or 4.

As used herein, “substantially pure” refers to a purity of at leastabout 80%. In some embodiments, the purity of a compound of the presentinvention has a purity of at least about 85%, about 90%, about 95%, orabout 99%. In a further embodiment, the purity of a compound of thepresent invention has a purity of at least about 99.5%, or about 99.8%.In an even further embodiment, the purity of a compound of the presentinvention has a purity of at least about 99.85%, about 99.90%, about99.94%, about 99.95%, or about 99.99%. In some embodiments, the compoundof the present invention is selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof. In some embodiments, the compound of the presentinvention is a polymorph. In some embodiments, the compound of thepresent invention is a polymorph of a compound according to Formula I.In some embodiments, the compound of the present invention is apolymorph of Compound 1.

The typical impurities that may be present in a compound of the presentinvention include one or more selected from the group consisting ofby-product, isomer, intermediate, and solvent. In some embodiments, theimpurities that may be present in a compound of the present invention isat most about 10%, about 8%, about 5%, about 2%, or about 1% relative tothe compound of Formula II. In a further embodiment, the impurities thatmay be present in a compound of the present invention is at most about0.5%, about 0.2%, about 0.15%, or about 0.1% relative to the compound ofFormula II. In an even further embodiment, the impurities that may bepresent in a compound of the present invention is at most about 0.05%,about 0.02%, or about 0.01% relative to the compound of Formula II. Insome embodiments, the substantially pure compound of Formula II have atmost about 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.15, 0.1, or 0parts per million (p.p.m.) of residual by-product or by-productsrelative to the compound of Formula II.

In some embodiments, the impurities include one or more by-productsselected from the group consisting of2-acetyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione,2,6-Diacetyl-naphtho[2,3-b]furan-4,9-dione,2,7-Diacetyl-naphtho[2,3-b]furan-4,9-dione,3-Acetyl-naphtho[2,3-b]furan-4,9-dione, Naphtho[2,3-b]furan-4,9-dione,Naphtho[2,3-b]furan-4,9-dione, Naphtho[2,3-b]furan-4,9-diol, and1-(4,9-Dihydroxy-naphtho[2,3-b]furan-2-yl)-ethanone.

In some embodiments, the impurities include manganese (Mn).

The purity of a compound of the present invention may be determined withvarious devices. In some embodiments, the purity is determined with HPLC(High Performance Liquid Chromatography). In some embodiments, thepurity is determined with NMR (Nuclear Magnetic Resonance). In a furtherembodiment, the purity is determined with HPLC and NMR.

These highly pure compositions containing Compound 1 exhibit asignificantly improved safety profile in animal experiments compared toless pure compositions that contain Compound 1. No signs of any adverseeffects of highly pure Compound 1 have been observed in mice. Inaddition, these highly pure compositions containing Compound 1 have beentested in patients and have demonstrated exceptional safety. Forexample, FIG. 13 illustrates the toxicity observed with a compositionwith about 90% purity for Compound 1, while FIG. 14 illustrates that thehighly pure compositions having about 95% or greater purity for Compound1 are safe and effective. In the study shown in FIG. 13,immunosuppressed mice with established subcutaneous FaDu human head andneck cancer (upper panel) or MDA-231 human breast cancer (lower panel)were given indicated amount of Compound 1, or vehicle control orally(po). Compound 1 was formulated in GELUCIRE™. All regimens wereadministered daily (qd). Body weights were evaluated periodically duringtreatment. Each point represents the mean±SEM of eight tumors.Significant toxicity was observed with about 90% pure Compound 1. Atotal of 4 mice died during the treatment in the first experiment (upperpanel) (one on day 16, 2 on day 19, and 1 on day 23), and their bodyweights were, therefore, not included in the plot after their death. Atotal of 3 mice died during the treatment in the second experiment(lower panel) (1 on day 14 and 2 on day 21), and their body weightswere, therefore, not included in the plot after their death. In thestudy shown in FIG. 14, immunosuppressed mice with establishedsubcutaneous FaDu human head and neck cancer (upper panel) or MDA-231human breast cancer (lower panel) were given indicated amount ofCompound 1, or vehicle control orally (po). Compound 1 was formulated inGELUCIRE™. All regimens were administered daily (qd). Body weights wereevaluated periodically during treatment. Each point represents themean±SEM of eight tumors. Compound 1 with higher purity waswell-tolerated and showed no signs of toxicity. All mice remainedhealthy throughout the treatment in both experiments. In a Phase Istudy, the dose of Compound 1 was escalated from 20 mg to 2000 mg/day,and a maximum tolerated dose (MTD) not reached. No dose-limitingtoxicity was observed. Patients tolerated Compound 1 very well withoutdrug-induced adverse effects, which is in sharp contrast to cancerchemotherapeutics. The clinical safety profile of the substantially purecompositions of Compound 1 is among the best for oncology drugs inhistory.

Pharmaceutical Formulations

Certain excipients or enhancers were found to enhance the oralbioavailability of particles of a compound according to Formula I of agiven particle size distribution in a pharmaceutical formulation. Forexample, the addition of the pharmaceutically compatible excipientGELUCIRE™ 44/14 (a polyethylene glycol glyceryl laurate produced byGattefossé) can increase the bioavailability of Compound 1 having amedian particle size of less than or equal to about 20 microns. Examplesof other excipients than can be used to enhance or control oralbioavailability include surfactants, such as TWEEN 80™ or TWEEN 20™ (apolysorbate, i.e., a polyoxyethylene sorbitan monolaurate) or certainlipids, such as phosphatidylcholines, e.g.,dimyristoylphosphatidylcholine (DMPC). Surfactants include compoundsthat are amphiphilic and contain both hydrophobic and hydrophilicgroups. Other excipients can include, for example, a glycerol ester of afatty acid, a glycerol ester of a saturated fatty acid, a glycerol esterof a saturated fatty acid having from 8 to 18 carbons, glyceryl laurate,polyethylene glycol, a polyoxyethylene sorbitan alkylate, cellulose orcellulose derivatives, such as microcrystalline cellulose andcarboxymethyl cellulose (CMC), as well as lipids, such as sterols, e.g.,cholesterol. Other excipients can include antioxidants, such as VitaminE. Other excipients and additional components can be included in apharmaceutical formulation according to the present invention, as willbe appreciated by one of skill in the art. For example, other activeagents, standard vehicles, carriers, liquid carriers, saline, aqueoussolutions, diluents, surface active agents, dispersing agents, inertdiluents, granulating and disintegrating agents, binding agents,lubricating agents, glidants, discharging agents, sweetening agents,flavoring agents, coloring agents, preservatives, physiologicallydegradable compositions such as gelatin, aqueous vehicles and solvents,oily vehicles and solvents, suspending agents, dispersing or wettingagents, suspending agents, emulsifying agents, demulcents, buffers,salts, thickening agents, gelatins, fillers, emulsifying agents,antioxidants, antibiotics, antifungal agents, stabilizing agents, water,glycols, oils, alcohols, crystallization retarding agents (e.g., toretard crystallization of a sugar), starches, sugars, sucrose, surfaceactive agents, agents to increase the solubility of any otheringredient, such as a polyhydroxy alcohol, for example glycerol orsorbitol, pharmaceutically acceptable polymeric or hydrophobicmaterials, and other components can be included. The appropriateadditional agent or agents to add will depend on the dosage form (e.g.,injectable solution, capsule, or pill), as will be appreciated by oneskilled in the art.

The compound according to Formula I of the present invention may beformulated into “pharmaceutical compositions”. Embodiments according tothe present invention include various dosage forms including a compound,which can be useful, for example, for treating a patient. For example,oral dosage forms can include a tablet, pill, capsule (hard or soft),caplet, powder, granule, suspension (e.g., in an aqueous or oilyvehicle), solution (e.g., in an aqueous or oily vehicle), gel, cachet,troche, lozenge, syrup, elixir, emulsion, draught, oil-in-wateremulsion, or a water-in-oil emulsion. Because of their ease inadministration, tablets and capsules may represent a preferred oraldosage. Solid oral dosage forms may be sugar coated or enteric coated bystandard techniques. For example, nasal and other mucosal sprayformulations (e.g. inhalable forms) can include purified aqueoussolutions of the active compounds with preservative agents and isotonicagents. Such formulations are preferably adjusted to a pH and isotonicstate compatible with the nasal or other mucous membranes.Alternatively, they can be in the form of finely divided solid powderssuspended in a gas carrier, of an inhalant, or of an aerosol. Suchformulations may be delivered by any suitable means or method, e.g., bynebulizer, atomizer, metered dose inhaler, or the like. For example, apharmaceutical composition according to the present invention may beadministered topically, for example, in the form of an ointment, cream,or suppository. For example, a pharmaceutical composition according tothe present invention may be administered by injecting an injectant.Thus, a dosage form according to the present invention can have, forexample, a solid, semi-solid, liquid, or gaseous form. Suitable dosageforms include but are not limited to oral, rectal, sub-lingual, mucosal,nasal, ophthalmic, subcutaneous, intramuscular, intravenous, parenteral,transdermal, spinal, intrathecal, intra-articular, intra-arterial,sub-arachinoid, bronchial, lymphatic, and intra-uterile administration,and other dosage forms for systemic delivery of active ingredients. Anactive ingredient, for example, a compound according to Formula I may becontained in a formulation that provides quick release, sustainedrelease, delayed release, or any other release profile known to oneskilled in the art after administration to a subject (patient). The modeof administration and dosage form selected for a given treatment isclosely related to the therapeutic amounts of the compounds orcompositions which are desirable and efficacious for the given treatmentapplication as well as factors such as the mental state and physicalcondition of the subject (patient).

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, as a plurality of single unitdoses, or in a multi-dose form. As used herein, a “unit dose” is adiscrete amount of the pharmaceutical composition including apredetermined amount of the active ingredient. The amount of the activeingredient in each unit dose is generally equal to the total amount ofthe active ingredient that would be administered or a convenientfraction of a total dosage amount such as, for example, one-half orone-third of such a dosage. A formulation of a pharmaceuticalcomposition of the invention suitable for oral administration may be inthe form of a discrete solid dosage unit. Each solid dosage unitcontains a predetermined amount of the active ingredient, for example aunit dose or fraction thereof. As used herein, an “oily” liquid is onewhich includes a carbon or silicon based liquid that is less polar thanwater. In such pharmaceutical dosage forms, the active agent preferablyis utilized together with one or more pharmaceutically acceptablecarrier(s) therefore and optionally any other therapeutic ingredients.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The compositions of the presentinvention can be provided in unit dosage form, wherein each dosage unit,e.g., a teaspoon, tablet, capsule, solution, or suppository, contains apredetermined amount of the active drug or prodrug, alone or inappropriate combination with other pharmaceutically-active agents. Theterm “unit dosage form” refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of the composition of the present invention,alone or in combination with other active agents, calculated in anamount sufficient to produce the desired effect.

Dosage forms of the present pharmaceutical composition can be preparedby techniques known in the art and contain a therapeutically effectiveamount of an active compound or ingredient. Any technique known orhereafter developed may be used for the preparation of pharmaceuticalcompositions or formulations according to the invention. In general,preparation includes bringing the active ingredient into associationwith a carrier or one or more other additional components, and then, ifnecessary or desirable, shaping or packaging the product into a desiredsingle- or multi-dose unit. Powdered and granular formulations accordingto the invention may be prepared using known methods or methods to bedeveloped. Such formulations may be administered directly to a subject,or used, for example, to form tablets, fill capsules, or prepare anaqueous or oily suspension or solution by addition of an aqueous or oilyvehicle thereto. A tablet may be made by compression or molding, or bywet granulation, optionally with one or more accessory ingredients.Compressed tablets may be prepared by compressing, in a suitable device,the active ingredient in a free-flowing form such as a powder orgranular preparation. Molded tablets may be made by molding, in asuitable device, a mixture of the active ingredient, a pharmaceuticallyacceptable carrier, and at least sufficient liquid to moisten themixture. Tablets may be non-coated, or they may be coated using methodsknown in the art or methods to be developed. Coated tablets may beformulated for delayed disintegration in the gastrointestinal tract of asubject, for example, by use of an enteric coating, thereby providingsustained release and absorption of the active ingredient. Tablets mayfurther include ingredients to provide a pharmaceutically elegant andpalatable preparation. Hard capsules including the active ingredient maybe made using a physiologically degradable composition, such as gelatin.Such hard capsules include the active ingredient. Soft gelatin capsulesincluding the active ingredient may be made using a physiologicallydegradable composition, such as gelatin. Such soft capsules include theactive ingredient, which may be mixed with water or an oil medium.Liquid formulations of a pharmaceutical composition of the inventionthat are suitable for administration may be prepared, packaged, and soldeither in liquid form or in the form of a dry product intended forreconstitution with water or another suitable vehicle prior to use.Liquid suspensions, in which the active ingredient is dispersed in anaqueous or oily vehicle, and liquid solutions, in which the activeingredient is dissolved in an aqueous or oily vehicle, may be preparedusing conventional methods or methods to be developed. Liquid suspensionof the active ingredient may be in an aqueous or oily vehicle. Liquidsolutions of the active ingredient may be in an aqueous or oily vehicle.To prepare such pharmaceutical dosage forms, an active ingredient, e.g.,a naphthofuran, can be intimately admixed with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration. In preparing the compositions inoral dosage form, any of the usual pharmaceutical media may be employed.

In some embodiments according to the present invention, an item ofmanufacture includes a container containing a therapeutically effectiveamount of a pharmaceutical composition including a compound according toFormula I. The container can include a pharmaceutically acceptableexcipient. The container can include printed labeling instructions. Forexample, the printed labeling can indicate the dosage and frequency withwhich the pharmaceutical composition should be administered, and whetherthe composition should be administered with food or within a definedperiod of time before or after ingestion of food. The composition can becontained in any suitable container capable of holding and dispensingthe dosage form that will not significantly interact with thecomposition. The labeling instructions can be consistent with themethods of treatment described herein. The labeling can be associatedwith the container by a means that maintains a physical proximity of thetwo. By way of non-limiting example, the container and the labeling mayboth be contained in a packaging material such as a box or plasticshrink wrap or may be associated with the instructions being bonded tothe container such as with glue that does not obscure the labelinginstructions or other bonding or holding means.

Processes for Making Pharmaceutical Formulations Having SelectedParticle Size Distribution and Identifying an Optimum Particle SizeDistribution Milling Processes

In a method according to the present invention, a milling or grindingprocess can be used to reduce the size of particles of an activeingredient or compound according to Formula I. For example, a milling orgrinding process can be suitable for producing particles having a mediansize of 200 μm, 150 μm, 100 μm, 40 μm, 20 μm, 5 μm, 2 μm or greater orlesser size. Such a milling or grinding process can include, forexample, ball milling, roll milling, jet milling, wet milling,ultrasonic milling, grinding, and combinations. For example, the processcan reduce particle size by impacting particles with a hard surface, orby subjecting the particles to high pressure, e.g., squeezing a particlebetween two surfaces. For example, in jet milling, a stream of gasentrains particles and accelerates them to high velocities. Theparticles then impact other particles and walls and fracture intosmaller particles. For example, in wet milling, particles are combinedwith a liquid, and the resultant slurry is passed through a high shearmixer to fracture the particles. For example, in ultrasonic milling,particles, for example, in a slurry, are exposed to ultrasonicradiation. Cavitation induced by the ultrasound can fracture theparticles into particles of smaller size.

It can be advantageous to lower the temperature of the particles priorto subjecting them to the milling or grinding operation. For example,the temperature can be lowered to a cryogenic temperature, e.g., byexposing the particles to or immersing the particles in a cryogenicfluid, such as liquid nitrogen. Such lowering of the temperature canrender the particles more brittle and more susceptible to having theirsize reduced in the milling or grinding operation. Subsequent to themilling or grinding process, a selection process, such as sieving, canbe used to narrow the range of particle sizes.

Crystallizing Process

Crystallization is the main separation and purification step for themanufacturing of drug substances. Crystallization can also be utilizedto control particle size. The particle size distribution (PSD) obtainedduring crystallization is influenced by a combination of variousmechanisms that occur during crystallization, such as nucleation,growth, aggregation, attrition, breakage, etc. Control of PSD duringcrystallization is critical to achieving the desired product properties.When the particle size cannot be consistently controlled duringcrystallization to meet the desired specifications, an extra processingstep such as dry milling can be included. (Braat, et al Crystallization:Particle Size Control, Encyclopedia of Pharmaceutical Technology: ThirdEdition, Published on 2 Oct. 2006)

Methods for Treatment of Cancer

A method according to the present invention for treating, delaying theprogression of, preventing a relapse of, alleviating a symptom of, orotherwise ameliorating a human, mammal, or animal subject afflicted witha neoplasm includes administering a therapeutically effective amount ofa pharmaceutical composition including particles of a predetermined sizedistribution, for example, a compound according to Formula I such asCompound 1, a pure compound, a pure product and/or a pure pharmaceuticalcomposition, so that the volume growth of the neoplasm is slowed, thevolume growth of the neoplasm is stopped, the neoplasm decreases involume, and/or a cancerous neoplasm is killed. A few examples of typesof neoplasms that may be amenable to treatment by this method includesolid tumors, malignant tumors, cancers, metastatic tumors, neoplasmsincluding cancer stem cells, neoplasms in which the STAT3 pathway isimplicated, carcinomas, and sarcomas. A non-exhaustive list of cancersthat may be amenable to treatment by administration of particles of acompound according to Formula I include the following: breast cancer,head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer,colorectal carcinoma, prostate cancer, melanoma, sarcoma, liver cancer,brain tumor, leukemia, multiple myeloma, gastric cancer, and lymphoma.The STAT3 pathway may be implicated in these cancers. A non-exhaustivelist of cancers that may be amenable to treatment by administration ofparticles of, for example, a compound according to Formula I include thefollowing: colorectal cancer, breast cancer, ovarian cancer, lungcancer, melanoma and medulloblastoma. The CSC pathway may be implicatedin these cancers. A non-exhaustive list of other cancers that may beamenable to treatment by administration of particles of, for example, acompound according to Formula I include the following: lung cancer,cervical cancer, renal cell carcinoma, hepatocellular carcinoma,esophageal cancer, glioma, bladder cancer, colorectal cancer, breastcancer, prostate cancer, pancreatic cancer, endometrial cancer, thyroidcancer, bile duct cancer, bone cancer, eye cancer (retinoblastoma),gallbladder cancer, pituitary cancer, rectal cancer, salivary glandcancer, and nasal pharyngeal cancer.

Cancer Stem Cells

In recent years, a new model of tumorigenesis has gained wideacceptance, where it is hypothesized that only a small fraction of theentire tumor mass are responsible for the tumorigenic activities withinthe tumor, whereas the old or clonal genetic model posits that all themutated tumor cells contribute equally to such tumorigenic activities.This small fraction of tumorigenic cells, according to the new model,are transformed cells with stem-cell-like qualities and are called“cancer stem cells” (CSCs). Bonnet and Dick first demonstrated, in vivo,the presence of CSCs in acute myeloid leukemia (AML) during the 1990s.Their data showed that only a small subpopulation of human AML cells hadthe ability to transfer AML when transplanted into immunodeficient micewhile other AML cells were incapable of inducing leukemia. Later, theseCSCs were shown to have the same cellular markers, CD34⁺/CD38⁻, asprimitive hematopoietic stem cells. (Bonnet, D., Normal and leukaemicstem cells. Br J Haematol, 2005. 130(4): p. 469-79). Since then,researchers have found CSCs conclusively in various types of tumorsincluding those of the brain, breast, skin, prostate, colorectal cancer,and so on.

The CSC model of tumorigenesis would explain why tens or hundreds ofthousands of tumor cells need to be injected into an experimental animalin order to establish a tumor transplant. In human AML, the frequency ofthese cells is less than 1 in 10,000. (Bonnet, D. and J. E. Dick, Humanacute myeloid leukemia is organized as a hierarchy that originates froma primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7). Eventhough rare within a given tumor cell population, there is mountingevidence that such cells exist in almost all tumor types. However, ascancer cell lines are selected from a sub-population of cancer cellsthat are specifically adapted to grow in tissue culture, the biologicaland functional properties of cancer cell lines can undergo dramaticchanges. Therefore, not all cancer cell lines contain CSCs.

Cancer stem cells share many similar traits with normal stem cells. Forexample, CSCs have self-renewal capacity, namely, the ability to giverise to additional tumorigenic cancer stem cells, typically at a slowerrate than other dividing tumor cells, as opposed to a limited number ofdivisions. CSCs also have the ability to differentiate into multiplecell types, which would explain histological evidence that not only manytumors contain multiple cell types native to the host organ, but alsothat heterogeneity is commonly retained in tumor metastases. CSCs havebeen demonstrated to be fundamentally responsible for tumorigenesis,cancer metastasis, and cancer reoccurrence. CSCs are also called tumorinitiating cells, cancer stem-like cells, stem-like cancer cells, highlytumorigenic cells, tumor stem cells, solid tumor stem cells, or supermalignant cells.

The existence of cancer stem cells has fundamental implications forfuture cancer treatments and therapies. These implications aremanifested in disease identification, selective drug targeting,prevention of cancer metastasis and recurrence, and development of newstrategies in fighting cancer.

The efficacy of current cancer treatments are, in the initial stages oftesting, often measured by the size of the tumor shrinkage, i.e., theamount of tumor mass that is killed off. As CSCs would form a very smallproportion of the tumor and have markedly different biologiccharacteristics than their more differentiated progenies, themeasurement of tumor mass may not necessarily select for drugs that actspecifically on the stem cells. In fact, cancer stem cells appear to beresistant to radiotherapy (XRT) and also refractory to chemotherapeuticand targeted drugs. (Hambardzumyan, D., M. Squatrito, and E. C. Holland,Radiation resistance and stem-like cells in brain tumors. Cancer Cell,2006. 10(6): p. 454-6; Baumann, M., M. Krause, and R. Hill, Exploringthe role of cancer stem cells in radioresistance. Nat Rev Cancer, 2008.8(7): p. 545-54; Ailles, L. E. and I. L. Weissman, Cancer stem cells insolid tumors. Curr Opin Biotechnol, 2007. 18(5): p. 460-6). Normalsomatic stem cells are naturally resistant to chemotherapeuticagents—they have various pumps (such as MDR) that pump out drugs, andDNA repair proteins. Further, they also have a slow rate of cellturnover while chemotherapeutic agents target rapidly replicating cells.Cancer stem cells, being the mutated counterparts of normal stem cells,may also have similar mechanisms that allow them to survive drugtherapies and radiation treatment. In other words, conventionalchemotherapies and radiotherapies kill differentiated or differentiatingcells, which form the bulk of the tumor that are unable to generate newhighly tumorigenic cancer stem cells. The population of cancer stemcells that gave rise to the differentiated and differentiating cells, onthe other hand, could remain untouched and cause a relapse of thedisease. A further danger for conventional anti-cancer therapy is thepossibility that chemotherapeutic treatment leaves onlychemotherapy-resistant cancer stem cells, and the ensuing recurrenttumor will likely also be resistant to chemotherapy.

Since the surviving cancer stem cells can repopulate the tumor and causerelapse, it is imperative that anti-cancer therapies include strategiesagainst CSCs (see FIG. 18). This is akin to eliminating the roots inorder to prevent dandelions from regrowth even if the weed's groundlevel mass has been cut. (Jones, R. J., W. H. Matsui, and B. D. Smith,Cancer stem cells: are we missing the target? J Natl Cancer Inst, 2004.96(8): p. 583-5). By selectively targeting cancer stem cells, it becomespossible to treat patients with aggressive, non-resectable tumors andrefractory or recurrent cancers, as well as preventing the tumormetastasis and recurrence. Development of specific therapies targetingcancer stem cells may improve survival and the quality of life of cancerpatients, especially for sufferers of metastatic cancers. The key tounlocking this untapped potential is the identification and validationof pathways that are selectively important for cancer stem cellself-renewal and survival. Unfortunately, though multiple pathwaysunderlying tumorigenesis in cancer or self-renewal in embryonic andadult stem cells have been elucidated in the past, very few pathwayshave been identified and validated for cancer stem cell self-renewal andsurvival.

There has also been a lot of research into the identification andisolation of cancer stem cells. Methods used mainly exploit the abilityof CSCs to efflux drugs, or are based on the expression of surfacemarkers associated with cancer stem cells.

For example, since CSCs are resistant to many chemotherapeutic agents,it is not surprising that CSCs almost ubiquitously overexpress drugefflux pumps such as ABCG2 (BCRP-1) (Ho, M. M., et al., Side populationin human lung cancer cell lines and tumors is enriched with stem-likecancer cells. Cancer Res, 2007. 67(10): p. 4827-33; Wang, J., et al.,Identification of cancer stem cell-like side population cells in humannasopharyngeal carcinoma cell line. Cancer Res, 2007. 67(8): p. 3716-24;Haraguchi, N., et al., Characterization of a side population of cancercells from human gastrointestinal system. Stem Cells, 2006. 24(3): p.506-13; Doyle, L. A. and D. D. Ross, Multidrug resistance mediated bythe breast cancer resistance protein BCRP (ABCG2). Oncogene, 2003.22(47): p. 7340-58; Alvi, A. J., et al., Functional and molecularcharacterisation of mammary side population cells. Breast Cancer Res,2003. 5(1): p. R₁-8), and other ATP binding cassette (ABC) superfamilymembers (Frank, N. Y., et al., ABCB5-mediated doxorubicin transport andchemoresistance in human malignant melanoma. Cancer Res, 2005. 65(10):p. 4320-33; Schatton, T., et al., Identification of cells initiatinghuman melanomas. Nature, 2008. 451(7176): p. 345-9). Accordingly, theside population (SP) technique, originally used to enrich hematopoieticand leukemic stem cells, was also employed to identify and isolate CSCs.(Kondo, T., T. Setoguchi, and T. Taga, Persistence of a smallsubpopulation of cancer stem-like cells in the C6 glioma cell line. ProcNatl Acad Sci USA, 2004. 101(3): p. 781-6). This technique, firstdescribed by Goodell et al., takes advantage of differential ABCtransporter-dependent efflux of fluorescent dyes such as Hoechst 33342to define and isolate a cell population enriched in CSCs (Doyle, L. A.and D. D. Ross, Multidrug resistance mediated by the breast cancerresistance protein BCRP (ABCG2). Oncogene, 2003. 22(47): p. 7340-58;Goodell, M. A., et al., Isolation and functional properties of murinehematopoietic stem cells that are replicating in vivo. J Exp Med, 1996.183(4): p. 1797-806). Specifically, the SP is revealed by blocking drugefflux with verapamil, at which point the dyes can no longer be pumpedout of the SP.

Researchers have also focused on finding specific markers thatdistinguish cancer stem cells from the bulk of the tumor. Most commonlyexpressed surface markers by the cancer stem cells include CD44, CD133,and CD166. (Collins, A. T., et al., Prospective identification oftumorigenic prostate cancer stem cells. Cancer Res, 2005. 65(23): p.10946-51; Li, C., et al., Identification of pancreatic cancer stemcells. Cancer Res, 2007. 67(3): p. 1030-7; Ma, S., et al.,Identification and characterization of tumorigenic liver cancerstem/progenitor cells. Gastroenterology, 2007. 132(7): p. 2542-56;Prince, M. E., et al., Identification of a subpopulation of cells withcancer stem cell properties in head and neck squamous cell carcinoma.Proc Natl Acad Sci USA, 2007. 104(3): p. 973-8; Ricci-Vitiani, L., etal., Identification and expansion of human colon-cancer-initiatingcells. Nature, 2007. 445(7123): p. 111-5; Singh, S. K., et al.,Identification of a cancer stem cell in human brain tumors. Cancer Res,2003. 63(18): p. 5821-8; Dalerba, P., et al., Phenotypiccharacterization of human colorectal cancer stem cells. Proc Natl AcadSci USA, 2007. 104(24): p. 10158-63). Sorting tumor cells basedprimarily upon the differential expression of these surface marker(s)have accounted for the majority of the highly tumorigenic CSCs describedto date. Therefore, these surface markers are well validated foridentification and isolation of cancer stem cells from the cancer celllines and from the bulk of tumor tissues.

Recent studies have uncovered the presence of cancer stem cells (CSCs)with an exclusive ability to regenerate tumors. These CSCs exist inalmost all tumor types and are functionally linked with continuedmalignant growth, cancer metastasis, recurrence, and cancer drugresistance. CSCs and their more differentiated progenies appear to havemarkedly different biologic characteristics. Conventional cancer drugscreenings depend on measurement of the amount of tumor mass, therefore,they may not necessarily select for drugs that act specifically on theCSCs. In fact, CSCs have been demonstrated to resistant to standardchemotherapies and radiotherapy, and to becoming enriched after standardanti-cancer treatments, which result in cancer refractory andrecurrence. Methods of isolating these cells include but not limited toidentification by their ability of efflux Hoechst 33342, identificationby the surface markers these cells express, such as CD133, CD44, CD166,and others, and enrichment by their tumorigenic property. The mountingevidence linking cancer stem cells to tumorigenesis unravel enormoustherapeutic opportunity of targeting cancer stem cells.

The data provided herein, combined with recent breakthroughs in CSCresearch, allows the present invention to provide an array of methodsdirected at inhibiting CSCs, methods directed at inhibiting both CSCsand heterogeneous cancer cells, and methods of treating cancers thathave CSCs in specific or cancers in general. The present invention alsoprovides related methods (e.g., manufacturing and drug candidatescreening), materials, compositions and kits. The method can prevent theCSCs from self-renewal, such that it is no longer able to replenish itsnumbers by dividing into tumorigenic CSC cells. Or, the method caninduce cell death in CSCs, or in both CSCs and heterogeneous cancercells.

This method can be used to treat a subject's cancer. Cancers that aregood candidates for such treatment include but are not limited to:breast cancer, head and neck cancer, lung cancer, ovarian cancer,pancreatic cancer, colorectal carcinoma, prostate cancer, renal cellcarcinoma, melanoma, hepatocellular carcinomas, cervical cancer,sarcomas, brain tumors, gastric cancers, multiple myeloma, leukemia, andlymphomas. In some embodiments, the method is used to treat livercancers, head and neck cancers, pancreatic cancers, and/or gastriccancers. In some embodiments, the method is used to treat multiplemyeloma, brain tumors, and sarcomas.

Further, as CSCs have been demonstrated to be fundamentally responsiblefor tumorigenesis, cancer metastasis and cancer reoccurrence, anymethods of the invention directed to inhibiting CSCs, or both CSCs andheterogeneous cancer cells, can be practiced to treat cancer that ismetastatic, refractory to a chemotherapy or radiotherapy, or hasrelapsed in the subject after an initial treatment.

In some embodiments, the cancer stem cell inhibitor according to thepresent invention is: a compound of Formula 1, Compound 1, a polymorphof Compound 1, a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1, apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 2, a polymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dionecharacterized by an X-ray diffraction pattern including two or morepeaks from a peak at least at about 10.2 degrees 2θ, a peak at least atabout 11.9 degrees 2θ, a peak at least at about 14.1 degrees 2θ, a peakat least at about 14.5 degrees 2θ, a peak at least at about 17.3 degrees2θ, a peak at least at about 22.2 degrees 2θ, and a peak at least atabout 28.1 degrees 2θ and any combinations thereof, a polymorph of2-acetyl-4H, 9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3, apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern including two or more peaks from a peakat least at about 7.5 degrees 2θ, a peak at least at about 9.9 degrees2θ, a peak at least at about 12.3 degrees 2θ, a peak at least at about15 degrees 2θ, a peak at least at about 23 degrees 2θ, a peak at leastat about 23.3 degrees 2θ, a peak at least at about 24.6 degrees 2θ, anda peak at least at about 28.4 degrees 2θ and any combinations thereof,2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof; a polymorph of a compound of Formula 1, Compound 1, apolymorph of Compound 1, a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1, apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 2, a polymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dionecharacterized by an X-ray diffraction pattern including two or morepeaks from a peak at least at about 10.2 degrees 2θ, a peak at least atabout 11.9 degrees 2θ, a peak at least at about 14.1 degrees 2θ, a peakat least at about 14.5 degrees 2θ, a peak at least at about 17.3 degrees2θ, a peak at least at about 22.2 degrees 2θ, and a peak at least atabout 28.1 degrees 2θ and any combinations thereof, a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3, apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern including two or more peaks from a peakat least at about 7.5 degrees 2θ, a peak at least at about 9.9 degrees2θ, a peak at least at about 12.3 degrees 2θ, a peak at least at about15 degrees 2θ, a peak at least at about 23 degrees 2θ, a peak at leastat about 23.3 degrees 2θ, a peak at least at about 24.6 degrees 2θ, anda peak at least at about 28.4 degrees 2θ and any combinations thereof,2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof; or a substantially pure form of a compound of Formula1, Compound 1, a polymorph of Compound 1, a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1, apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 2, a polymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dionecharacterized by an X-ray diffraction pattern including two or morepeaks from a peak at least at about 10.2 degrees 2θ, a peak at least atabout 11.9 degrees 2θ, a peak at least at about 14.1 degrees 2θ, a peakat least at about 14.5 degrees 2θ, a peak at least at about 17.3 degrees2θ, a peak at least at about 22.2 degrees 2θ, and a peak at least atabout 28.1 degrees 2θ and any combinations thereof, a polymorph of2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 3, apolymorph of 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione characterizedby an X-ray diffraction pattern including two or more peaks from a peakat least at about 7.5 degrees 2θ, a peak at least at about 9.9 degrees2θ, a peak at least at about 12.3 degrees 2θ, a peak at least at about15 degrees 2θ, a peak at least at about 23 degrees 2θ, a peak at leastat about 23.3 degrees 2θ, a peak at least at about 24.6 degrees 2θ, anda peak at least at about 28.4 degrees 2θ and any combinations thereof,2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof; a particle form of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof (also referred to herein as the “Compound of theInvention”).

The present invention provides a method of identifying a drug candidatecapable of inhibiting a cancer stem cell. In some embodiments, the drugcandidate is capable of inducing cell death in CSC or at leastinhibiting its self-renewal. In a further embodiment, the drug candidateis capable of inducing cell death in CSC or at least inhibiting itsself-renewal, and inducing cell death in heterogeneous cancer cells.Various phases in the pathway can be targeted for screening the drugcandidate.

Accordingly, in another aspect, the Compound of the Invention can beused to formulate a pharmaceutical composition to treat or preventdisorders or conditions. Some of the disorders include but are notlimited to: autoimmune diseases, inflammatory diseases, inflammatorybowel diseases, arthritis, autoimmune demyelination disorder,Alzheimer's disease, stroke, ischemia reperfusion injury and multiplesclerosis. Some of the disorders are cancers and include but are notlimited to: various types of breast cancers, head and neck cancers, lungcancers, ovarian cancers, pancreatic cancers, colorectal carcinoma,prostate cancers, renal cell carcinoma, melanoma, hepatocellularcarcinomas, cervical cancers, sarcomas, brain tumors, gastric cancers,multiple myeloma, leukemia, and lymphomas.

Accordingly, in an aspect, the present invention provides a method ofinhibiting cancer stem cells where an effective amount of the Compoundof the Invention is administered to the cells. Cancers known to haveCSCs are good candidates for such treatments, and include but are notlimited to: various types of breast cancers, head and neck cancers, lungcancers, ovarian cancers, pancreatic cancers, colorectal adenocarcinoma,prostate cancers, liver cancers, melanoma, multiple myeloma, braintumors, sarcomas, medulloblastoma, and leukemia.

Further, as CSCs have been demonstrated to be fundamentally responsiblefor tumorigenesis, cancer metastasis and cancer reoccurrence, anymethods of the invention directed to inhibiting CSCs can be practiced totreat cancer that is metastatic, refractory to a chemotherapy orradiotherapy, or has relapsed in the subject after an initial treatment.

In some embodiments of the method, the cancer being treated is selectedfrom the following group: liver cancer, colon cancer, head and neckcancer, pancreatic cancer, gastric cancer, renal cancer, sarcoma,multiple myeloma, metastatic breast cancer, metastatic prostate cancer,leukemia, lymphoma, pancreatic esophageal cancer, brain tumor, glioma,bladder cancer, endometrial cancer, thyroid cancer, bile duct cancer,bone cancer, eye cancer (retinoblastoma), gallbladder cancer, pituitarycancer, rectal cancer, salivary gland cancer, and nasal pharyngealcancer.

In an aspect, the present invention provides a method of treating cancerin a subject, where a therapeutically effective amount of apharmaceutical composition including the Compound of the Invention isadministered to the subject. The cancer may be metastatic. The subjectmay be a mammal, e.g., a human being.

Treatment by administration of particles of, for example, a compoundaccording to Formula I to a subject (patient) suffering from a neoplasmmay be indicated for the following conditions. The neoplasm may berefractory to treatment by chemotherapy, radiotherapy, or hormonetherapy. The neoplasm may not be amenable to surgical resection. Theneoplasm may have relapsed in the subject (patient). Cancer stem cellshave been implicated in the relapse of neoplasms; killing the cancerstem cells or inhibiting their self-renewal by a method according to thepresent invention may prevent the neoplasm from regenerating itself.Treatment by administration of particles of naphthofuran may slow orstop the volume growth of a neoplasm or decrease the volume of aneoplasm by, for example, inducing the death of, inhibiting the growthand/or division of, and/or selectively killing neoplastic cells. Forexample, a treatment according to the present invention may induce celldeath of a cell of the neoplasm. For example, the treatment may act toinhibit the STAT3 pathway of a neoplastic cell.

Treatment by administration of particles of, for example, a Compound ofthe Invention to a subject (patient) suffering from a neoplasm may beused to prevent relapse of a neoplasm and/or as an adjuvant therapy tosurgical resection.

A pharmaceutical composition including particles of, for example, aCompound of the Invention may be administered orally, as this is aconvenient form of treatment. For example, the pharmaceuticalcomposition may be administered orally no more than four times per day.Alternatively, the pharmaceutical composition can be administeredintravenously or intraperitoneally.

In a method according to the present invention, the therapeuticallyeffective amount of the pharmaceutical composition including particles,polymorphs and/or purified forms of a Compound of the Invention can be atotal daily dose in the range of from about 20 mg to about 2000 mg,about 100 mg to about 1500 mg, about 160 mg to about 1400 mg, or fromabout 180 mg to 1200 mg. In some embodiments, the therapeuticallyeffective amount of the pharmaceutical composition including particles,polymorphs and/or purified forms of a Compound of the Invention is atotal daily dose in the range of from about 200 mg to about 1500 mg, orfrom about 360 mg to 1200 mg. In some embodiments, the therapeuticallyeffective amount of the pharmaceutical composition including particles,polymorphs and/or purified forms of a Compound of the Invention is atotal daily dose in the range of from about 400 mg to about 1000 mg. Insome embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is a total daily dose ofabout 1000 mg.

In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in asingle daily dose. For example, in some embodiments, the therapeuticallyeffective amount of the pharmaceutical composition including particles,polymorphs and/or purified forms of a Compound of the Invention isadministered in a single daily dose in a range of from about 20 mg QD toabout 2000 mg QD. In some embodiments, the therapeutically effectiveamount of the pharmaceutical composition including particles, polymorphsand/or purified forms of a Compound of the Invention is administered ina single daily dose in a range of about 20 mg QD to about 1000 mg QD.

In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in morethan one daily dose. For example, in some embodiments, thetherapeutically effective amount of the pharmaceutical compositionincluding particles, polymorphs and/or purified forms of a Compound ofthe Invention is administered in two daily doses, where the total dailydose is in a range of from about 160 mg to 1400 mg. In some embodiments,the therapeutically effective amount of the pharmaceutical compositionincluding particles, polymorphs and/or purified forms of a Compound ofthe Invention is administered in two daily doses, where the total dailydose is in a range of from about 320 mg to 1200 mg. In some embodiments,the therapeutically effective amount of the pharmaceutical compositionincluding particles, polymorphs and/or purified forms of a Compound ofthe Invention is administered in two daily doses, where the total dailydose is in a range of from about 400 mg to 1000 mg. In some embodiments,the therapeutically effective amount of the pharmaceutical compositionincluding particles, polymorphs and/or purified forms of a Compound ofthe Invention is administered in two daily doses, where the total dailydose is about 1000 mg.

In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in twodaily doses, where each dose is in a range of from about 80 mg to 1000mg. In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in twodaily doses, where each dose is in a range of from about 160 mg to 600mg. In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in twodaily doses, where each dose is in a range of from about 200 mg to about500 mg. In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in twodaily doses, where each dose is about 500 mg.

In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in threedaily doses, where the total daily dose is in a range of from about 240mg to about 1500 mg. In some embodiments, the therapeutically effectiveamount of the pharmaceutical composition including particles, polymorphsand/or purified forms of a Compound of the Invention is administered inthree daily doses, where the total daily dose is in a range of fromabout 480 mg to about 1500 mg.

In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in threedaily doses, where each dose is in a range of from about 80 mg to 500mg. In some embodiments, the therapeutically effective amount of thepharmaceutical composition including particles, polymorphs and/orpurified forms of a Compound of the Invention is administered in threedaily doses, where each dose is in a range of from 160 mg to 500 mg.

A Compound of the Invention or a pharmaceutical composition thereof canbe administered through any one of or through a combination of routes,for example, orally, intravenously, or intraperitoneally. For example,in some embodiments, a Compound of the Invention can be administeredorally. In some embodiments, a Compound of the Invention can beadministered orally in a formulation that includes Gelucire and Tween80.

A Compound of the Invention can be administered in a dose to achieve ablood concentration in a subject, e.g., a patient, of compound in therange of from at least about 0.002 μM to about 30 μM for a time of atleast 2 hours to no more than 24 hours. In some embodiments, a Compoundof the Invention can be administered in a dose to achieve a bloodconcentration in a subject of compound in the range of from at leastabout 0.2 μM to about 1 μM for a time of at least 2 hours to no morethan 24 hours. equals to or above about 0.2 μM, 0.5 μM, 1.0 μM, 1.5 μM,2.0 μM, 2.5 μM, 3.0 μM 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM,10.0 μM, 15.0 μM for at least 2 hours and less than 24 hours. In someembodiments, a Compound of the Invention can be administered in a doseto achieve a blood concentration in a subject of compound equals to orabove about 1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, 5.0 μM, 10.0 μM, 15.0 μM forat least 2 hours and less than 24 hours. In some embodiments, a Compoundof the Invention can be administered in a dose to achieve a bloodconcentration in a subject of compound equals to or above about 2.0 μM,3.0 μM, 5.0 μM, 10.0 μM for at least 2 hours and less than 24 hours. Insome embodiments, a Compound of the Invention can be administered in adose to achieve a blood concentration in a subject of compound equals toor above about 3.0 μM, or 5.0 μM for at least 2 hours and less than 24hours.

A Compound of the Invention can be administered in a dose to achieve ablood concentration in a subject, e.g., a patient, of compound in therange of from at least about 0.002 μM·h to about 300 μM·h in 24 hours.In some embodiments, a Compound of the Invention can be administered ina dose to achieve area under the curve in 24 hours (AUC24) in a subjectequals to or above about 0.2 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM,3.0 μM 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 15.0 μMfor at least 2 hours and less than 24 hours. In some embodiments, aCompound of the Invention can be administered in a dose to achieve ablood concentration in a subject of compound equals to or above about1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, 5.0 μM, 10.0 μM, 15.0 μM for at least 2hours and less than 24 hours. In some embodiments, a Compound of theInvention can be administered in a dose to achieve a blood concentrationin a subject of compound equals to or above about 2.0 μM, 3.0 μM, 5.0μM, 10.0 μM for at least 2 hours and less than 24 hours. In someembodiments, a Compound of the Invention can be administered in a doseto achieve a blood concentration in a subject of compound equals to orabove about 3.0 μM, or 5.0 μM for at least 2 hours and less than 24hours. In some embodiments, a Compound of the Invention can beadministered in a dose to achieve area under the curve in 24 hours(AUC_(0-24 hr)) in a subject equals to or above about 2 μM*hr, 10 μM*hr,20 μM*hr, 30 μM*hr, 40 μM*hr, 50 μM*hr, 60 μM*hr, 70 μM*hr, 80 μM*hr, 90μM*hr, 100 μM*hr, 125 μM*hr, 150 μM*hr, 200 μM*hr, 250 μM*hr, 300 μM*hr,400 μM*hr, and 500 μM*hr.

If the condition of the subject (patient) so requires, doses of thepharmaceutical composition may be administered as a continuous orpulsatile infusion. The duration of a treatment may be decades, years,months, weeks, or days, as long as the benefits persist. The foregoingranges are provided only as guidelines and are subject to optimization.

In a method according to the invention, cells of the neoplasm areselectively killed by administering the pharmaceutical composition, sothat the blood molar concentration of the compound is at least aneffective concentration and less than a harmful concentration for afirst continuous time period that is at least as long as an effectivetime period and shorter than a harmful time period. The blood molarconcentration can be less than the effective concentration after thefirst continuous time period. The effective concentration can be aconcentration sufficiently high, so that neoplastic cells, e.g., cancercells, are killed. The effective time period can be sufficiently long,so that neoplastic cells, e.g., cancer cells, are killed. The harmfulconcentration can be a concentration at which normal cells are damagedor killed. The harmful time period can be a time period sufficientlylong for normal cells to be damaged or killed. For example, theeffective concentration can be equal to or above about 0.02 μM, about0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 1 μM, about 3μM, about 10 μM or about 20 μM. For example, the non-harmfulconcentration can be equal to or below about 3 μM, about 10 μM, about 14μM, about 30 μM, or about 100 μM. For example, the effective time periodcan be equal to or above about 2 hour, about 4 hours, about 6 hours,about 12 hours, about 24 hours, or about 48 hours. For example, toachieve non-harmful exposure for normal cells, drug concentration ofCompound 1 has to be substantially cleared from blood within about 12hours, about 24 hours. “Substantially clearance from blood” means blooddrug concentration decrease by at least about 50%, at least about 60%,at least about 80%, at least about 90%. For example, an effectiveconcentration can be a concentration that exceeds the IC₅₀ of cancercells when the compound is administered for some time period. Forexample, an effective time period can be a time period over which cancercells are selectively inhibited or killed when the compound isadministered at least at the effective concentration. For example, aharmful concentration can be a concentration that exceeds the IC₅₀ ofnormal cells when the compound is administered for any time period. Forexample, a harmful time period can be a time period over which normal aswell as cancer cells are inhibited or killed when the compound isadministered at the effective concentration.

One of skill in the art can administer the pharmaceutical composition byselecting dosage amount and frequency so as to achieve a hereindescribed “selective pharmacokinetic profile” (SPP) deemed necessary forselective killing neoplastic cells, such as cancer cells, and sparingnormal cells. Such consideration of the SPP can also guide the design ofthe pharmaceutical composition, for example, the particle sizedistribution and distribution of shapes of the particles.

In a method according to the invention, the pharmaceutical compositionis administered orally in a dosage form such as a tablet, pill, capsule(hard or soft), caplet, powder, granule, suspension, solution, gel,cachet, troche, lozenge, syrup, elixir, emulsion, oil-in-water emulsion,water-in-oil emulsion, or draught.

Identifying an Optimum Particle Size Distribution

In a method according to the invention, an optimum particle sizedistribution of a compound according to Formula I, Compound 1, apolymorph of Compound 1, and/or a substantially pure form of Compound 1for treating a human, mammal, or animal afflicted with a neoplasm can bedetermined as follows. At least one set of particles including thecompound can be prepared. In preparing the set of particles, forexample, the particle size of a sample of solid compound can be reducedby, for example, dissolving the compound and nebulizing the solution,dissolving the compound and sonicating the solution, ball milling thesolid compound, roll milling the solid compound, grinding the solidcompound, and/or sieving the solid compound. The particle sizedistribution of the at least one set of particles can be determined by amethod or combination of methods known to one of skill in the art. Forexample, the particle size distribution can be determined using atechnique such as sieve analysis, optical microscopic counting, electronmicrograph counting, electroresistance counting, sedimentation time,laser diffraction, acoustic spectroscopy, another technique, or acombination of techniques. The at least one set of particles can beadministered to neoplastic cells and to normal cells at a predeterminedconcentration and for a predetermined period of time. The effect of theparticles on the metabolism, division, and/or other indicator of thevitality of the neoplastic cells and the normal cells can be observed.The observed effect of the particles on the neoplastic cells can be usedto assign an effectivity rating to each set of particles. For example, aset of particles that inhibits the metabolism and/or division of theneoplastic cells, damages or kills the neoplastic cells, or otherwiseexhibits high antitumor activity can be assigned a high effectivityrating. The observed effect of the particles on the normal cells can beused to assign a toxicity rating to each set of particles. For example,a set of particles that inhibits the metabolism and/or division of thenormal cells or damages or kills the normal cells or where the normalcells otherwise exhibit a low tolerability of the set of particles canbe assigned a high toxicity rating.

For example, the set of particles can be administered to neoplasticcells and normal cells in vitro. For example, the effectivity rating canbe equal to, proportional to, or a monotonically increasing function ofthe IC₅₀ of the neoplastic cells. For example, the toxicity rating canbe equal to, proportional to, or a monotonically increasing function ofthe IC₅₀ of the normal cells.

For example, the set of particles can be administered to neoplasticcells and normal cells in vivo in a test animal. For example, the testanimal can be a mammal, primate, mouse, rat, guinea pig, rabbit, or dog.For example, the effectivity rating can be equal to, proportional to, ora monotonically increasing function of the decrease in volume of theneoplastic cells following administration of the set of particles. Forexample, the toxicity rating can be equal to, proportional to, or amonotonically increasing function of the decrease in mass of the testanimal following administration of the set of particles. For example,the set of particles can be administered to a human in a clinical study.A method of treating a neoplasm can include administering atherapeutically effective amount of a set of particles of the compoundaccording to Formula I, Compound 1, a polymorph of Compound 1, and/or asubstantially pure form of Compound 1 to a human, mammal, or animalafflicted with the neoplasm. Prior to administration of the particles ofthe compound, the compound according to Formula I, Compound 1, apolymorph of Compound 1, and/or a substantially pure form of Compound 1to an animal or a human or to cells in vitro, the particles can besuspended in a pharmaceutically acceptable excipient.

The effectivity rating and/or the toxicity rating of each set ofparticles having a first particle size distribution can be compared withthe effectivity rating and/or the toxicity rating of another set or setsof particles having a particle size distribution different than thefirst particle size distribution. A set of particles of a compound thathas a high effectivity rating and a low toxicity rating can be effectivein inhibiting or killing neoplastic, e.g., cancer, cells, but sparenormal cells. One of skill in the art can select as an optimum set theset of particles having an effectivity rating greater than, a toxicityrating less than, and/or a weighted effectivity rating and toxicityrating sum greater than the at least one other set of particles (forexample, the effectivity rating can be weighted with a positivecoefficient and the toxicity rating can be weighted with a negativecoefficient). One of skill the art can also use another criteria toselect the optimum set of particles, for example, particles having a sumof the weighted effectivity rating and the weighted ratio of theeffectivity rating over the toxicity rating. The particle sizedistribution of the optimum set of particles can be considered anoptimum particle size distribution for the compound tested. The optimumparticle size distribution may be different for one compound, e.g.,Compound 1, than for another compound, e.g., a compound according toFormula I that is not Compound 1. The optimum particle size distributionfor a given compound may differ when determined by administration tocells in vitro, to a small test animal, and to a large test animal.However, the optimum particle size distribution determined byadministration of a given compound to an organism in vitro or in vivomay represent a rational starting point for optimizing the particle sizedistribution for another compound or for administration to anotherorganism.

An optimum set of particles of the compound according to Formula I,Compound 1, a polymorph of Compound 1, and/or a substantially pure formof Compound 1 can be included in the composition for reducing orinhibiting the replication or spread of neoplastic cells.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Example 1 Preparation of a Naphthofuran Compound

The procedure for preparation of a naphthofuran compound(2-acetylnaphtho[2,3-b]furan-4,9-dione) is summarized as follows:

Step 1: Bromination

To a 2 liter 3 neck round bottom flask equipped with a mechanicalstirrer, thermometer, and addition funnel is charged 3-butene-2-one(451.2 grams). To the addition funnel is added bromine (936.0 grams).After the content in the flask is cooled to −5° C., the bromine isdropped into the flask with vigorous stirring and maintainingtemperature at −5° C. over 30 minutes. The mixture is stirred for anadditional 15 minutes at −5° C., and then is split into 4 equalportions.

Step 2 Debromination

Each portion of the mixture along with tetrahydrofuran (2133.6 grams) isloaded into a 22 liter 4 neck round bottom flask equipped with amechanical stirrer, thermometer, and addition funnel. To the additionfunnel is charged DBU (1,3-Diazabicyclo[5.4.0]undec-7-ene, 222.9 grams).The DBU is dropped into the flask with vigorous stirring and maintainingtemperature at 0° C.-5° C. over 30 minutes. The mixture is stirred foran additional 15 min at 0° C.-5° C.

Step 3: Coupling Reaction

2-hydroxy-1,4-naphthofuran (231 grams) is then added into the flask.Additional DBU (246.0 grams) is charged into the addition funnel andthen dropped into the mixture in the flask at such a rate that thetemperature of the reaction mixture does not exceed 40° C. After theaddition of DBU is complete, the resulting mixture is stirred overnightat room temperature, and a sample of the reaction mixture is taken forHPLC analysis.

Step 4: Crystallization

To the reaction mixture, water (10.8 liters) is charged, and theresulting mixture is cooled to 0° C.-3° C. for at least 30 minutes, thenfiltered via vacuum filter. The filtered solid is rinsed with 5% aqueoussodium bicarbonate (3 liters), water (3 liters), 1% aqueous acetic acid(3 liters) and ethanol twice (2×1 liter) successively.

The rinsed solid is stored and pooled together from other batches. Thecombined crude product (28.73 kg) is loaded along with ethyl acetate(811.7 kg) into a 500 gallon vessel equipped with a mechanical stirrer,thermometer, and a condenser. Under nitrogen atmosphere, the mixture isheated to reflux (72° C.) for 2 hours, and then filtered with a 10micron cartridge filter containing an active carbon layer to removeinsolubles.

Fresh hot ethyl acetate (10 kg) is used to rinse the vessel, transferline and filter. The combined filtrate is cooled to 0-5° C. and held atthis temperature for 2 hours, and then is filtered with 20 inch Buchnerfilter. The filtered solid product is rinsed with 0-5° C. ethyl acetate(5.7 kg), and dried under vacuum at 40° C. to a constant weight. Theremaining filtrate is reduced in volume by 63% by evaporation, and thecrystallization process was repeated again to generate a second crop ofproduct which was also dried under the same condition as the first cropof product.

A lot of the naphthofuran compound obtained following the procedure. Thepurity for the lot of the compound is 95.44 area % (HPLC).

Example 2 Preparation of a Naphthofuran Compound

Another procedure for the preparation of a naphthofuran compound(2-acetylnaphtho[2,3-b]furan-4,9-dione) is summarized as follows:

Step 1: Bromination

A 12 L RBF (Round Bottom Flask)(protected from light with UV filters)was charged with MVK (2,160 ml, 26.4 mol) and cooled to −9.6° C. in adry-ice/acetone bath. Bromine (1,300 ml, 25.3 mol) was added slowly,over 2 hrs and 20 min, maintaining T=<−2.6° C. (T_(max)). The resultingyellow mixture was stirred for additional 28 min.

Step 2: De-Hydrobromination

A 72 L RBF with pre-cooled THF (Tetrahydrofuran) (20 L, 5 ml/g HNQ(2-Hydroxy-1,4-naphtoquinone)) was charged with brominated product fromthe above and the resulting solution was cooled to −4.8° C. DBU (4,200ml, 28.1 mol) dissolved in THF (4,200 ml) was added slowly, over 2 hrsand 20 min, maintaining T<0.3° C.(T_(max)). The resulting suspension wasstirred for 42 min.

Step 3: Coupling

2-Hydroxy-1,4-naphthofuran (4,003 g, 23.0 mol) was charged, in oneportion, into the reaction mixture from the above, at −1.8° C. A coolingbath was added while a second portion of DBU (3,780 ml, 25.3 mol) wasadded over 48 minutes to bring the reaction temperature to 40° C. Thecooling bath was removed and the reaction mixture was stirred over theweekend, open to the air.

Step 4: Isolation of Crude Material

A 200 L reactor with pre-cooled water (100 L, 25 ml/g HNQ) was chargedwith the reaction mixture from the above. The resulting suspension wascooled to 6.0° C., and then stirred at T=3±3° C. for ˜1 hour. Theresulting suspension was then filtered, and the collected solids weretransferred back to the 200 L reactor.

After stirring in 5% NaHCO₃ aqueous (26 L, 6.5 ml/g HNQ) for 1 hour, thesuspension was filtered. The collected solids were transferred back tothe 200 L reactor, stirred in water (26 L) for 1 hour, and thenfiltered.

The wet solids were transferred back to the 200 L reactor, stirred in 1%aqueous acetic acid (26 L) for ˜1 hour, filtered and then washed on thefilter funnel with water (10 L). The collected solids were transferredback to the 200 L reactor and heated in ethanol (17.5 L; 4.3 ml/g HNQ)to a gentle reflux (77.4° C.). The resulting suspension was cooled to4.2° C. and filtered.

The wet solids were transferred to a 100 L reactor and heated in ethanol(17.5 L; 4.3 ml/g HNQ) to a reflux (77.6° C.). The resulting suspensionwas cooled to 4.5° C. and filtered. The wet cake was de-liquoredovernight. ¹H NMR and HPLC samples were taken. ¹H NMR: Compound 1/NDHF(2-acetyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione) 42:58%; HPLC:Compound 1/NDHF 74:11 area %.

The solids were dried in a vacuum oven at 50° C., over 4 days, affording2,268 g of crude Compound 1. ¹H NMR: Compound 1/NDHF 41:59%; HPLC:Compound 1/NDHF 67:11 area %.

Step 5: Oxidation of the Naphthodihydrofurane

The crude Compound 1 (2.268 kg) was slurried in toluene (77 L). MnO₂(9536 g) was added and the mixture was heated to a gentle reflux. TLC(1:1 EA:hexane) showed complete reaction after 1 hour.

The reaction mixture was then filtered hot through a preheated pad ofCelite (1530 g, bottom layer), activated charcoal (2230 g, middlelayer), and Celite (932 g, top layer). The yellow-orange filtrate wascollected.

The filtrate was concentrated on the rotovap to approximately 1/10volume. The slurry was filtered and washed with toluene. The crystalswere then dried at 50° C. to give 952 g (42%) of dark yellow solid.HPLC: 99.94%. ¹H NMR showed no naphthodihydrofuran.

The crystals were dried at 50° C. under vacuum for an additional 46-65hours to reduce the amount of residual toluene in the material.

Step 6: Ethyl Acetate Treatment

The Compound 1 (5816 g) was charged to a 200 L reaction vessel. Ethylacetate (145 L, 25 mL/g) was added, and the solution was heated toreflux over 2 hours 26 minutes. Reflux was maintained for 5 hours 30minutes, and the mixture was then cooled and maintained overnight to 17°C.

The slurry was filtered on a polyethylene frit. The yellow crystals wereair dried, then placed in trays in a vacuum oven for 75 hours, giving5532 g (95.1% yield) of yellow solids. HPLC: 99.86%. ¹H NMR matches thestructure of Compound 1.

Step 7: Ethyl Acetate Re-Crystallization

A 2 L RBF was charged with crude material (10 g) and ethyl acetate (900ml). The mixture was refluxed at ˜77° C. and then more ethyl acetate(100 ml) was added to achieve complete dissolution. The resultingclear-yellowish solution was stirred at reflux for ˜30 minutes, and thenthe heating was removed. The mixture was stirred overnight at roomtemperature.

The resulting suspension was filtered and the collected yellow solidswere rinsed on the funnel with ethyl acetate (30 ml). The wet solid wasdried in vacuum oven at 40-50° C., over 4 hours, to obtain 8.53 g ofyellow crystalline product (total yield ˜17%).

¹H NMR: consistent with structure; HPLC: 99.94 area %; DSC: 228.68° C.,151 J/g.

Unless specifically indicated otherwise, Compound 1 used in thefollowing examples was prepared as in Example 1.

Example 3 Micronization of Naphthofuran Compound

For example, Compound 1 crystals were milled and passed through a 160micron (μm) sieve (Sieve #100, 150 μm opening) to generate the crystalsof approximately 160 microns or less.

For example, Compound 1 crystals were milled (The Retsch Ultracentrifugal Mill ZM 200; Single pass, at 18,000 rpm using 0.25 mmscreen) to a median particle size of about 20 micron. Table 3 presentsthe resultant distribution of particle sizes (Malvern 2000 with theHydro 2000S wet accessory). The columns present the maximum size ofparticles in the cumulative percent total presented in the subscript atthe header of the column. For example, the column D₉₀ presents the sizefor which 90% of the particles have an equal or lesser size. The columnD₅₀ represents the median size—half of the particles have a greatersize, and half of the particles have an equal or lesser size.

TABLE 3 Particle Size Distribution of Milled Compound 1. Particle Size(microns) D₉₀ D₅₀ D₁₀ Sample B 48.9 20.2 2.3

For example, Compound 1 crystals were micronized using a jet millingmethod (4″ Jet Mill, Venturi pressure=40, Mill pressure=100, Feedrate=1304 g/hour) to a median particle size of about 2 micron, aspresented in Table 4. Particle size analysis was performed using a dryparticle method (Sympatec Helos/KF Particle Size Analyzer).

TABLE 4 Particle Size Distribution of Micronized Compound 1 ParticleSize (microns) D₉₀ D₅₀ D₁₀ Sample A 4.63 2.07 0.53

A cumulative distribution function derived from a log-normal model ofparticle size distribution provided a good fit to the data presented inTable 4. The cumulative distribution function was represented as

${{{CDF}(d)} = {\frac{1}{2}\left( {1 + {{erf}\left( \frac{{\ln (d)} - {\ln \left( d_{median} \right)}}{\sigma \sqrt{2}} \right)}} \right)}},$

where erf is the error function, d is the particle diameter variable,d_(median) is the median particle size, and σ is a parameter related tothe breadth of the cumulative distribution function. CDF(d) representsthe fraction of particles having a size less than or equal to d. Settingd_(median) to the observed median of 2.07 micron, fitting of the modelyielded a value of σ=1.06. The model indicated a mean diameter of 3.6micron and a mode diameter of 0.67 micron. The model also suggests aspecific area of the particles of 2200 m²/kg, although this does notaccount for factors such as surface roughness.

Example 4 Pharmacokinetics in Mice of 2 Micron, 20 Micron, 150 MicronMedian Particle Size Formulations

In an experiment, micronized Compound 1 prepared in step 6 of Example 2with a mean particle size of 2 micron, 20 microns, 150 microns wereformulated as suspensions in 20% Gelucire 44/14 and 1% Tween 80 andadministered orally to mice at 100 mg/kg. Each time point represents theaverage of 3 mice (FIG. 16).

As shown in FIG. 16, while the Compound 1 with a particle size ofbetween 125-150 micron shows a lower level of exposure compared to the 2micron and 20 micron particles when all are dosed at 100 mg/kg, it showsthe same pattern. Compound 1 particle sizes of 20 micron (d50) showsimilar plasma exposure in mice as dose Compound 1 with particle sizedof 2 micron (d50). Furthermore, if you double the exposure of the125-150 micron Compound 1, it would be very similar to the 2 and 20micron PK graph.

Example 5 Formulations Having Reduced Particle Size Exhibit GreaterInhibition of Tumor Growth

In the present studies, Compound 1 shows no or weak efficacy if itadministered to mice in a composition with particle size greater than 20micron. However, Compound 1 was found to have potent anti-tumor activitywith no observed toxicity if the compound is administered in acomposition of a particle size less than 5 micron.

In an experiment, a formulation of Compound 1 particles sieved to 160micron was tested in the model of immunosuppressed mice with establishedsubcutaneous xenograft FaDu human head and neck cancer. Thepharmaceutical composition was formulated as 80 mg/ml in 9% Gelucire,20% Vitamin E TPGS (Table 3). No efficacy was observed at the dose of400 mg/kg daily oral dosing (a vehicle control was also administered),as shown in FIG. 15. This dose level is 4 fold higher that that used inthe PK experiment shown in FIG. 16. Therefore these mice receive 4×higher exposure than that see by dosing 100 mg/kg 2 micron Compound 1which shows good efficacy. All regimens were administered daily (qd).

In an experiment, Compound 1 crystals were milled to a median particlesize of about 20 micron. Only weak or moderate efficacy was observedwhen the Compound 1 milled to a median particle size of about 20 micronswas dosed orally daily at 200 mg/kg in mice with xenografted FaDu humanhead and neck tumors (FIG. 15) (a vehicle control was alsoadministered). All regimens were administered daily (qd).

Compound 1 crystals prepared in Example 1 were also tested. The Compound1 crystals were micronized using a jet milling method (4″ Jet Mill,Venturi pressure=40, Mill pressure=100, Feed rate=1304 g/hour) to amedian particle size of about 2 micron, as presented in Table 4.

FaDu human head and neck cancer cells were inoculated subcutaneouslyinto female athymic nude mice (6×10⁶ cells/mouse) and allowed to formpalpable tumors. When the tumors reached approximately 100 mm³, theanimals were treated orally (po) with Compound 1 at 100 mg/kg or vehiclecontrol daily. Compound 1 was formulated at 10 mg/ml in 20% gelucire.Tumors and bodyweights were measured throughout treatment (FIG. 15).

Compound 1 was also micronized using a jet milling method (8″ PancakeMill, Ventury pressure=40, Mill pressure=40, Feed rate=1920 g/hour) to amedian particle size of about 2 micron, as presented in Table 5.Particle size analysis was performed using a dry particle method(Sympatec Helos/KF Particle Size Analyzer). Similar anti-tumor activitywas observed as the 2 micron material in Table 4.

TABLE 5 Particle Size Distribution of Micronized Compound 1 ParticleSize (microns) D₉₀ D₅₀ D₁₀ Sample A 5.5 2.21 0.51

Therefore, while Compound 1 of either 150 micron or 20 microns shows asimilar plasma exposure pattern as Compound 1 of 2 microns (FIG. 16).They show different efficacy: Compound 1 of 150 microns shows noefficacy (FIG. 15); Compound 1 of 20 microns shows weak or moderateefficacy; and Compound 1 of 2 microns shows strong efficacy.

As shown in FIG. 16, Compound 1 particle sizes of 20 micron (d50) showssimilar plasma exposure in mice as dose Compound 1 with particle sizedof 2 micron (d50). Surprisingly, however, Compound 1 with 20 micronparticle size shows only weak or moderate efficacy in mouse xenograftmodels, while Compound 1 with 2 micron particle size shows potentefficacy. This is an unexpected result as the common understanding isthat the efficacy of a drug is based on its pharmacokinetics. Thereforesince both particle sizes show the same pharmacokinetics, they shouldboth be equally efficacious.

Furthermore, if the exposure of the 125-150 micron Compound 1 isdoubled, it would be very similar to the 2 and 20 micron PK graph.Interestingly, when 150 micron Compound 1 is dosed to mice at a level ashigh as 400 mg/kg, it also shows no efficacy in xenograft models (FIG.15).

These results go against the conventional view of the reduction ofparticle size leading to increased plasma exposure and therefore betterefficacy.

Example 6 HPLC Assay

This HPLC method is to assess purity of naphthofuran, e.g.,2-acetylnaphtho[2,3-b]furan-4,9-dione (Compound 1), and its reactioncompletion by HPLC. All components will be expressed in area percent ofthe total peaks within the chromatogram.

TABLE 6A 1. APPARATUS AND MATERIALS Apparatus HPLC system with UVdetector and integration system Column Phenomenex Luna C18(2) 5-μm,4.6-mm × 250-mm (P/N 00G-4252-E0) or equivalent pH meter calibrated theday of use Acetonitrile HPLC Grade Dimethylsulfoxide (DMSO) ACS Grade orbetter Phosphoric acid ACS reagent Potassium phosphate, dibasic ACSreagent Compound 1 Reference Material

2. Solution Preparations 10 mM Phosphate Buffer

Weigh 1.74 g of Potassium Phosphate, dibasic and dilute with 1 L ofPurified Water (adjust weights and volumes for amount needed). Adjustthe pH with Phosphoric Acid to pH 6.8.

Mobile Phase A

Prepare Mobile Phase A by mixing the 10 mM phosphate buffer andacetonitrile to a 80:20 buffer:acetonitrile ratio. Degas.

Mobile Phase B

Prepare Mobile Phase B by mixing the 10 mM phosphate buffer andacetonitrile to a 20:80 buffer:acetonitrile ratio. Degas.

Diluent

Mobile Phase A will be used as the diluent for all sample and standardpreparations.

3. Standards Preparations

Compound 1 Stock Standard (Concentration ≈1.0 mg/mL)

It will be prepared weighing 10 mg of Compound 1 Reference material intoa 20 mL scintillation vial; record weight±0.01 mg. Add 10 mL of DMSO andsonicate until the solids dissolve.

${Concentration} = \frac{\begin{matrix}{\left( {{{{Wt}.\mspace{14mu} {Reference}}\mspace{14mu} {Standard}},{mg}} \right) \times} \\{{Standard}\mspace{14mu} {Decimal}\mspace{14mu} {Purity}}\end{matrix}}{\left( {{{Volume}\mspace{14mu} {of}\mspace{14mu} {Stock}\mspace{14mu} {Solution}},{mL}} \right)}$

Stock Test Samples (Concentration ≈1.0 mg/mL)

Test Solutions will be prepared by weighing 10 mg of sample in a 20 mLscintillation vial and diluting with 10 mL of DMSO.

${Concentration} = \frac{\left( {{{Wt}.\mspace{14mu} {Sample}},{mg}} \right)}{\left( {{{Volume}\mspace{14mu} {of}\mspace{14mu} {Stock}\mspace{14mu} {Solution}},{mL}} \right)}$

Working Test Samples (Concentration ≈0.01 mg/mL)

This solution is prepared by transferring 1 mL into a 100 mL volumetricflask and diluting with diluent solution.

${Concentration} = \frac{\begin{matrix}{{Stock}\mspace{14mu} {Test}\mspace{14mu} {Sample}\mspace{14mu} {Concentration} \times} \\\left( {{{volume}\mspace{14mu} {transferred}},{mL}} \right)\end{matrix}}{\left( {{{Volume}\mspace{14mu} {of}\mspace{14mu} {Working}\mspace{14mu} {Solution}},{mL}} \right)}$

TABLE 6B 4. INSTRUMENT OPERATING CONDITIONS Flow Rate 0.8 mL/min. Columntemp 30° C. Detector Wavelength 270 nm Injection Volume 40 μL GradientProfile 0-5 min - 0% B to 0% B 5-19 min - 0% B to 90% B 19-24 min - 90%B to 90% B 24-29 min - 90% B to 0% B  Note: 5 min equilibration timebetween injections at 100% A Run Time 29 min

5. Operating Procedure

Inject solutions in the following sequence:

1. Diluent blank (1×)

2. Compound 1 Working Standard (5×)

3. Test Solutions (2× each)

4. Working Standards (1× each)

6. System Suitability

The system is suitable for use if the following criteria are met.

-   1. Diluent blank injection at the beginning of the sequence contains    no interfering peaks with any identified impurities-   2. The initial, 5 replicate injections of the Compound 1 working    standard have (1) % RSD_(peak area) <3.0%; (2) %    RSD_(retention time) <3.0%; and (3) mean tailing factor <2.0.-   3. In the chromatogram for the bracketed standard, (1) retention    time is 97.0-103.0% of the mean retention time from the initial    suitability injections and (2) its area % is 97.0-103.0% of the    initial value.

7. Calculations

All peaks will be reported as area % of the total peaks in thechromatogram, this will be calculated by the integration software by wayof the following formula:

${{Area}\mspace{14mu} \%} = {\frac{{Area}\mspace{14mu} {counts}\mspace{14mu} {of}\mspace{14mu} {peak}}{{Total}\mspace{14mu} {area}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {peaks}} \times 100}$

NMR and TLC

TABLE 6C NMR Apparatus Varian Inova 500 NMR Spectrometer Pulse SequenceS2pul Solvent CDC13 Temp. 25.0° C./298.1 K Relax delay 1.000 sec Pulse45.0 degrees Acq. time 2.732 sec Width 11992.2 Hz 32 repetitions OBSERVEH1 499.7029706 MHz FT size 65536 Total time 1 min, 50 sec

TABLE 6D TLC on silica gel eluent ethyl acetate:hexane, 1:1visualization UV Rf₄₀₁ ~0.7 Rf_(NDHF) ~0.6

Example 7 Preparation of 2-acetylnaphtho[2,3-b]furan-4,9-dione

A procedure for the preparation of Compound 1 is provided below.

Step 1: Bromination

To a 2 liter 3 neck round bottom flask equipped with a mechanicalstirrer, thermometer, and addition funnel is charged 3-butene-2-one(451.2 grams). To the addition funnel is added bromine (936.0 grams).After the content in the flask is cooled to −5° C., the bromine isdropped into the flask with vigorous stirring and maintainingtemperature at −5° C. over 30 minutes. The mixture is stirred for anadditional 15 minutes at −5° C., and then is split into 4 equalportions.

Step 2: Debromination

Each portion of the mixture along with tetrahydrofuran (2133.6 grams) isloaded into a 22 liter 4 neck round bottom flask equipped with amechanical stirrer, thermometer, and addition funnel. To the additionfunnel is charged DBU (1,3-Diazabicyclo[5.4.0]undec-7-ene, 222.9 grams).The DBU is dropped into the flask with vigorous stirring and maintainingtemperature at 0° C.-5° C. over 30 minutes. The mixture is stirred foran additional 15 min at 0° C.-5° C.

Step 3: Coupling Reaction

2-hydroxy-1,4-naphthoquinone (231 grams) is then added into the flask.Additional DBU (246.0 grams) is charged into the addition funnel andthen dropped into the mixture in the flask at such a rate that thetemperature of the reaction mixture does not exceed 40° C. After theaddition of DBU is complete, the resulting mixture is stirred overnightat room temperature, and a sample of the reaction mixture is taken forHPLC analysis.

To the reaction mixture, water (10.8 liters) is charged, and theresulting mixture is cooled to 0° C.-3° C. for at least 30 minutes, thenfiltered via vacuum filter. The filtered solid is rinsed with 5% aqueoussodium bicarbonate (3 liters), water (3 liters), 1% aqueous acetic acid(3 liters) and ethanol twice (2×1 liter) successively.

Step 4: Crystallization

The rinsed solid is stored and pooled together from other batches. Thecombined crude product (28.73 kg) is loaded along with ethyl acetate(811.7 kg) into a 500 gallon vessel equipped with a mechanical stirrer,thermometer, and a condenser. Under nitrogen atmosphere, the mixture isheated to reflux (72° C.) for 2 hours, and then filtered with a 10micron cartridge filter containing an active carbon layer to removeinsolubles.

Fresh hot ethyl acetate (10 kg) is used to rinse the vessel, transferline and filter. The combined filtrate is cooled to 0-5° C. and held atthis temperature for 2 hours, and then is filtered with 20 inch Buchnerfilter. The filtered solid product is rinsed with 0-5° C. ethyl acetate(5.7 kg), and dried under vacuum at 40° C. to a constant weight.

The remaining filtrate is reduced in volume by 63% by evaporation, andthe crystallization process was repeated again to generate a second cropof product which was also dried under the same condition as the firstcrop of product.

Two lots of Compound 1 were obtained following the procedure. One lothas a purity of 91.64 area % and the other lot has a purity of 95.44area %, measured by HPLC.

Example 8 Preparation of Crude 2-acetylnaphtho[2,3-b]furan-4,9-dione

Another procedure for the preparation of Compound 1 is summarized asfollows.

Step 1: Bromination

A 12 L RBF (Round Bottom Flask) (protected from light with UV filters)was charged with MVK (2,160 ml, 26.4 mol) and cooled to −9.6° C. in adry-ice/acetone bath. Bromine (1,300 ml, 25.3 mol) was added slowly,over 2 hrs and 20 min, maintaining T=<−2.6° C. (T_(max)). The resultingyellow mixture was stirred for additional 28 min.

Step 2: De-Hydrobromination

A 72 L RBF with pre-cooled THF (Tetrahydrofuran) (20 L, 5 ml/g HNQ(2-Hydroxy-1,4-naphtoquinone)) was charged with brominated product fromthe above and the resulting solution was cooled to −4.8° C. DBU (4,200ml, 28.1 mol) dissolved in THF (4,200 ml) was added slowly, over 2 hrsand 20 min, maintaining T<0.3° C. (T_(max)). The resulting suspensionwas stirred for 42 min.

Step 3: Coupling

2-Hydroxy-1,4-naphthoquinone (4,003 g, 23.0 mol) was charged, in oneportion, into the reaction mixture from the above, at −1.8° C. A coolingbath was added while a second portion of DBU (3,780 ml, 25.3 mol) wasadded over 48 minutes to bring the reaction temperature to 40° C. Thecooling bath was removed and the reaction mixture was stirred over theweekend, open to the air.

Step 4: Isolation of Crude Material

A 200 L reactor with pre-cooled water (100 L, 25 ml/g HNQ) was chargedwith the reaction mixture from the above. The resulting suspension wascooled to 6.0° C., and then stirred at T=3±3° C. for ˜1 hour. Theresulting suspension was then filtered, and the collected solids weretransferred back to the 200 L reactor.

After stirring in 5% NaHCO₃ aqueous (26 L, 6.5 ml/g HNQ) for 1 hour, thesuspension was filtered. The collected solids were transferred back tothe 200 L reactor, stirred in water (26 L) for 1 hour, and thenfiltered.

The wet solids were transferred back to the 200 L reactor, stirred in 1%aqueous acetic acid (26 L) for ˜1 hour, filtered and then washed on thefilter funnel with water (10 L). The collected solids were transferredback to the 200 L reactor and heated in ethanol (17.5 L; 4.3 ml/g HNQ)to a gentle reflux (77.4° C.). The resulting suspension was cooled to4.2° C. and filtered.

The wet solids were transferred to a 100 L reactor and heated in ethanol(17.5 L; 4.3 ml/g HNQ) to a reflux (77.6° C.). The resulting suspensionwas cooled to 4.5° C. and filtered. The wet cake was de-liquoredovernight. ¹H NMR and HPLC samples were taken. ¹H NMR: Compound 1/NDHF(2-acetyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione) 42:58%; HPLC:Compound 1/NDHF 74:11 area %.

The solids were dried in a vacuum oven at 50° C., over 4 days, affording2,268 g of crude Compound 1. ¹H NMR: Compound 1/NDHF 41:59%; HPLC:Compound 1/NDHF 67:11 area %.

Example 9 Oxidation of the Naphthodihydrofurane

The crude Compound 1 (2.268 kg) was slurried in toluene (77 L). MnO₂(9536 g) was added and the mixture was heated to a gentle reflux. TLC(1:1 EA:hexane) showed complete reaction after 1 hour.

The reaction mixture was then filtered hot through a preheated pad ofCelite (1530 g, bottom layer), activated charcoal (2230 g, middlelayer), and Celite (932 g, top layer). The yellow-orange filtrate wascollected.

The filtrate was concentrated on the rotovap to approximately 1/10volume. The slurry was filtered and washed with toluene. The crystalswere then dried at 50° C. to give 952 g (42%) of dark yellow solid.HPLC: 99.94%. ¹H NMR showed no naphthodihydrofuran.

The crystals were dried at 50° C. under vacuum for an additional 46-65hours to reduce the amount of residual toluene in the material.

Example 10 Ethyl Acetate Treatment

The Compound 1 (5816 g) was charged to a 200 L reaction vessel. Ethylacetate (145 L, 25 mL/g) was added, and the solution was heated toreflux over 2 hours 26 minutes. Reflux was maintained for 5 hours 30minutes, and the mixture was then cooled and maintained overnight to 17°C.

The slurry was filtered on a polyethylene frit. The yellow crystals wereair dried, then placed in trays in a vacuum oven for 75 hours, giving5532 g (95.1% yield) of yellow solids. HPLC: 99.86%. ¹H NMR matches thestructure of Compound 1.

Example 11 Ethyl Acetate Re-Crystallization

A 2 L RBF was charged with crude material (10 g) and ethyl acetate (900ml). The mixture was refluxed at ˜77° C. and then more ethyl acetate(100 ml) was added to achieve complete dissolution. The resultingclear-yellowish solution was stirred at reflux for ˜30 minutes, and thenthe heating was removed. The mixture was stirred overnight at roomtemperature.

The resulting suspension was filtered and the collected yellow solidswere rinsed on the funnel with ethyl acetate (30 ml). The wet solid wasdried in vacuum oven at 40-50° C., over 4 hours, to obtain 8.53 g ofyellow crystalline product (total yield ˜17%).

¹H NMR: consistent with structure; HPLC: 99.94 area %; DSC: 228.68° C.,151 J/g.

Example 12 Identification of Naphthofuran Compounds that Target Cancerand Cancer Stem Cells Methods

In Life Evaluations:

Daily examinations into the health status of each animal were alsoconducted. Body weights were checked every three days. Food and waterwas supplied daily according to the animal husbandry procedures of thefacility. Treatment producing >20% lethality and or >20% net body weightloss were considered toxic. Results are expressed as mean tumor volume(mm³)±SE. P Values <0.05 are considered to be statistically relevant.

Animal Husbandry:

Male or female athymic nude mice 4-5 weeks (Charles River Laboratories,Wilmington, Mass.), were acclimated to the animal housing facility forat least 1 week before study initiation. All of the experimentalprocedures utilized were consistent with the guidelines outlined by theAmerican Physiology Society and the Guide for the Care and Use ofLaboratory Animals and were also approved by the Institutional AnimalCare and Use Committee of Boston Biomedical Inc. The animals were housedin groups of four in wood chip bedded cages in a room having controlledtemperature (68° F.-72° F.), light (12-h light-dark cycle), and humidity(45-55%). The animals were allowed free access to water and food duringthe experiment.

Example 13 Clinical Trial: Safety and Efficacy

2-acetylnaphtho[2,3-b]furan-4,9-dione was chosen to enter Phase Iclinical trial after receiving IND approval from US FDA and HealthCanada, which was a dose escalation study in adult patients withadvanced cancer who had failed standard therapies. A cycle consists oftwice-daily oral administration of the compound for 4 weeks. Cycles wererepeated every 4 weeks (28 days) until progression of disease,unacceptable toxicity, or another discontinuation criterion is met. Thedose escalation trial was conducted as open label and multicenter trial.A modified Simon accelerated titration scheme was used for doseescalation.

The primary objective of the trial was to determine the safety,tolerability, and recommended phase II dose (RP2D). The secondaryobjectives of the trial were to determine the pharmacokinetic profile ofthe compound, pharmacodynamics of the compound, and preliminaryantitumor activity of the compound.

The inclusion criteria included histologically or cytologicallyconfirmed solid tumor that is metastatic, unresectable, or recurrent;≧18 years of age; Measurable disease by RECIST; and Karnofsky ≧70%. Theexclusion criteria included chemotherapy, radiotherapy, immunotherapy orinvestigational agent within 4 weeks of first dose; surgery within 4weeks of first dose; and known brain metastases.

As of Feb. 7, 2011, 42 cancer patients with various advanced solidtumors who have failed chemotherapies were enrolled in the study. Thedemographics and baseline disease characteristics of the patientsselected under above criteria were summarized in Table 7.

TABLE 7 Demographics and Baseline Disease Characteristics Patients (N =42) Age (years) Mean 59.6 (12.7) Min, Max 28, 91 Sex [N (%)] Male 29(70.7%) Female 12 (29.3%) Race [N (%)] Causasian 33 (80.5%) Asian 3(7.3%) Black 1 (2.4%) Other 2 (4.9%) Hispanic 0 (0%) Prior Therapies¹ >320 2 2 1 4

Of those 42 patients, 10 cohorts were assessed at doses ranging from 20mg to 2000 mg/day. The dose escalation was well tolerated and no doselimiting toxicity was observed. Adverse events were generally mild withthe most common being: diarrhea, nausea, and fatigue. Grade 3 or greaterevents include: fatigue and diarrhea. These adverse events arerecordings on what these late stage cancer patients experience while onthe clinical trial, which may or may not be related to Compound 1. Theadverse events were summarized in Table 8

TABLE 8 Summary of Adverse Events Any Grade Grade 1 Grade 2 Grade 3Event Term # of Events % of Total # of Events % of Total # of Events %of Total # of Events % of Total Diarrhea 23 28.4% 20 24.7% 2 2.5% 2 2.5%Vomiting 14 17.3% 13 16.0% 1 1.2% 0 0.0% Nausea 10 12.3% 8 9.9% 2 2.5% 00.0% abdominal cramps 6 7.4% 5 6.2% 1 1.2% 0 0.0% weakness 5 6.2% 2 2.5%3 3.7% 0 0.0% Fatigue 4 4.9% 1 1.2% 2 2.5% 1 1.2% Anorexia 4 4.9% 3 3.7%1 1.2% 0 0.0% Dysgusia 3 3.7% 3 3.7% 0 0.0% 0 0.0% decreased appetite 22.5% 1 1.2% 1 1.2% 0 0.0% Fever 2 2.5% 1 1.2% 1 1.2% 0 0.0% Skin Rash 22.5% 2 2.5% 0 0.0% 0 0.0% dizzyness 2 2.5% 2 2.5% 0 0.0% 0 0.0% Loosestools 2 2.5% 2 2.5% 0 0.0% 0 0.0% Urine Color Change 2 2.5% 2 2.5% 00.0% 0 0.0%

To date, neither MTD nor RP2D has been reached. Doses through about 1000mg/day of the compound exhibited favorable pharmacokinetics withapparent linear pharmacokinetics and no evidence of drug accumulationupon repeated daily dosing every 28 days. At the 320 mg/day dose level,the plasma concentration of the compound was sustained for over 8 hoursat a concentration of at least 1.5 μM (IC₅₀ of the compound in vitro:30-500 nM). The mean plasma concentrations of different dose groups wereshown in FIG. 12.

Of the 42 patients dosed, 24 were evaluable for tumor response as ofFeb. 7, 2011; 16 (16/24 evaluable patients) achieved stable disease (8to 75+ weeks). The patients enrolled to date were summarized in Table 9.

TABLE 9 Patients Enrolled To Date

16/24 evaluable patients show SD/MR with 12 showing prolonged SD (>12weeks) by RECIST 1.1; New metastatic lesions were prevented in 83% ofpatients dosed.

The complete regression of a colon cancer metastatic lesion to kidney inpatient 0001 is shown in FIG. 19. In the 20 mg daily administration, ahigh concentration of the compound in urine of the patient was observed.The enrichment of the compound in urine (Table 10) explains the observedcomplete regression at relative low dosage.

The complete regression of a colon cancer metastatic lesion to kidney inpatient 0001 is shown in FIG. 19. In the 20 mg daily administration, ahigh concentration of the compound in urine of the patient was observed.The enrichment of the compound in urine might help explain the observedcomplete regression at relative low dosage.

TABLE 10 Compound 1 is Present at High Concentration in Patient UrineTotal Daily Time Post BBI608 Patient Dose (mg) Dose (min) (uM) 7 320120-240 4.3 360-480 23.1 8 320 120-240 7.9 360-480 1.8 9 320 120-240 8.9360-480 23.6 10 320 120-240 22.7 360-480 26.2 11 320 120-240 1.8 360-4804.5 12 400 120-240 4.11 360-480 3.86 14 400 120-240 1.42 360-480 5 15600 120-240 3.1 360-480 10.65 17 600 120-240 1.66 360-480 45.35 18 600120-240 2.41 360-480 6.3 20 800 120-240 6.17 360-480 118.25 21 800120-240 0.42 360-480 7.42 23 800 120-240 2.51 360-480 11.97

Accordingly, the compound showed an excellent safety profile. No doselimiting toxicity was observed to date.

A favorable PK profile with oral bid dosing was also observed. Theplasma concentration reached several folds over efficaciousconcentration (in vitro IC₅₀). AUC data is shown in Table 11.

TABLE 11 AUC summary for different dose levels Total daily dose (mg)AUC₀₋₂₄ BID dosing (uM*hr) SD 80 7.95 160 9.52 0.91 320 29.79 14.95 40053.61 19.55 600 27.27 5.97 800 26.43 5.27 1000 42.61 8.94 1400 28.383.95 2000 39.09 18.66

Moreover, signs of anti-tumor activity were observed. 16 out of 24patients showed SD/MR by RECIST in a range of tumors that are refractoryto chemotherapies, including colorectal adenocarcinoma, head and neckcancer, lung cancer, breast cancer, gastric cancer, and ovarian cancer,melanoma. There was one complete regression of a colon cancer metastaticlesion to kidney (FIG. 19). Patients treated with Compound 1 exhibited adramatic lack of new metastatic tumor lesions. Out of 24 evaluablepatients with advanced refractory cancers, over 80% showed no metastatictumors.

The patients achieved prolong stable disease (>16 weeks) during BBI608treatment were found to have high levels of p-STAT3 in their tumortissues prior to the treatment by immunohistochemistry usinganti-p-STAT3 antibody (FIG. 25).

Example 14 Dosing Regimens

The therapeutically effective amount of the pharmaceutical compositionincluding particles, polymorphs and/or purified forms of a Compound ofthe Invention can be a total daily dose in the range of from about 20 mgto about 2000 mg, from about 240 mg to about 1500 mg, or from about 400mg to about 1000 mg.

Suitable dosing regimens include administering particles, polymorphsand/or purified forms of a Compound of the Invention in a single dailydose. For example, the particles, polymorphs and/or purified forms of aCompound of the Invention are administered in a single daily dose in arange of from about 20 mg QD to about 1000 mg QD.

Suitable dosing regimens include administering particles, polymorphsand/or purified forms of a Compound of the Invention in more than onedaily dose. For example, the particles, polymorphs and/or purified formsof a Compound of the Invention are administered in two daily doses,where the total daily dose is in a range of from about 40 mg to about2000 mg. For example, the particles, polymorphs and/or purified forms ofa Compound of the Invention are administered in two daily doses, whereeach dose is in a range of from about 20 mg to 1000 mg. For example, theparticles, polymorphs and/or purified forms of a Compound of theInvention are administered in two daily doses, where each dose is in arange of from about 160 mg to 600 mg. For example, the particles,polymorphs and/or purified forms of a Compound of the Invention areadministered in two daily doses, where each dose is in a range of fromabout 200 mg to 500 mg. For example, the particles, polymorphs and/orpurified forms of a Compound of the Invention are administered in twodaily doses, where each dose is about 500 mg.

Suitable dosing regimens include administering particles, polymorphsand/or purified forms of a Compound of the Invention in three dailydoses, where the total daily dose is in a range of from about 60 mg toabout 1500 mg. For example, the particles, polymorphs and/or purifiedforms of a Compound of the Invention are administered in three dailydoses, where each dose is in a range of from about 20 mg to 500 mg. Forexample, the particles, polymorphs and/or purified forms of a Compoundof the Invention are administered in three daily doses, where each doseis in a range of from 160 mg to 500 mg.

The dosing regimen in which human subjects received about 500 mg ofCompound 1 twice daily (i.e., 1000 mg total daily dose) has shownachievement of best selective pharmacokinetics in almost all patientstreated. This dosing regimen, which is referred to herein as 500 mg BID,has demonstrated the desired pharmacokinetic properties of Compound 1 inhumans (FIG. 20).

In another suitable dosing regimen, 500 mg of Compound 1 areadministered three times a day (TID) to human subjects. While the levelof exposure of Compound 1 is not significantly improved by three times aday dosing as compared to twice daily dosing, the TID dosing doesincrease the exposure time of the drug in humans. This dose regimen,referred to herein as 500 mg TID, has shown good tolerability in humanswith no significant drug related adverse events observed.

In yet another suitable dosing regimen, at about or above 20 mg ofCompound 1 is administered once daily to human subjects. This dosingregimen, referred to herein as 20 mg QD, has shown therapeuticallyactive levels in patients, but is rapidly cleared from the blood inhumans (FIG. 21). This dose regimen has shown good tolerability inhumans and signs of potent antitumor activity in a colon cancer lesionin Kidney due to very high concentration of the drug in urine.

In yet another suitable dosing regimen, Compound 1 is administered withmilk with empty stomach which gives desirable pharmacokinetics (Table12).

TABLE 12 Effect of Milk on Compound 1 Pharmacokinetics PK ParameterFasting with Milk Fold Change Cmax (uM) 2.01 3.05 1.52 AUC_(0-24 hrs)20.12 31.40 1.56 Cmax (uM) 2.55 2.89 1.13 AUC_(0-24 hrs) 20.72 32.161.55

In yet another suitable dosing regimen, Compound 1 is administered withfood which delays the Tmax (Table 13).

TABLE 13 Taking Compound 1 with Food Causes a Delay in Tmax Tmax (hr)Patient Fasting With Milk With Food 20 2 2 8 21 6 6 6 22 8 8 10 24 — 6.310 27 — 0.5 6 28 — 6 10

Example 15 Naphthofuran Compounds Prolong Progression Free Survival

Prolongation of progression free survival (PFS) has been shown inpatients with advanced colorectal cancer which are refractory tochemotherapy (FIG. 22). Prolongation of progression free survival hasalso been seen in patients with head and neck cancer, gastric cancer,ovarian cancer, triple negative breast cancer, melanoma, adrenocorticoidcancer, and lung cancer.

Blood drug concentration of Compound 1 above 1 uM correlated with anincrease in progression free survival (FIG. 23) in patients with diversecancers including colorectal, gastric, head and neck, melanoma,chondrosarcoma, lung, prostate, ovarian, adrenocorticoid andangiosarcoma.

Example 16 Pharmacokinetic Profile of Compound 1

Compound 1 was found to be equally toxic to cancer cells and normalcells, and was concluded to be no potential for treating cancer (K.Hirai K. et al., Cancer Detection and Prevention, 23(6) (1999) 539-550;Takano A. et al., Anticancer Research 29:455-464, 2009). The studiesdescribed herein discovered counter-intuitively that cancer cells andcancer stem cells requires much shorter exposure than normal cells to bekilled by Compound 1. Normal cells can tolerate up to 24 hours ofexposure to Compound 1. Furthermore, the studies here discovered thatnormal cells can recover after a short period of no-drug exposure, whilecancer cells cannot recover once they are exposed to a certainconcentration of the Compound 1 for at least 2 hours. Based on thesestudies, a special pharmacokinetic exposure [termed selectivepharmacokinetic profile (SPP), or preferred pharmacokinetic profile(PPP), which is used interchangeably in this publication] was designedfor Compound 1 using the data shown below in Table 14 for achievingselective antitumor activity in patients (FIG. 24).

TABLE 14 Use of particle size to achieve preferred pharmacokinetic (PK)exposure for increasing plasma drug concentration and reducing toxicityto normal cells Compound 608 IC50 (uM) Normal Cells Treatment CD34⁺ BMCD34⁺ BM Cancer Cells Time Erythroid Myeloid PMBCs DU145 HT29 4-12 h<0.2 <0.5 12-24 h  >30 >30 14 <0.2 <0.5  72 h 3

Suitable SPP or PPP exposure to a Compound of the Invention such asCompound 1, particles, polymorphs and/or purified forms thereof, is atleast or above 1.0 μM for at least 2 hours, and blood drug concentrationhas to be cleared substantially within 24 hours.

For example, the patient maintains an exposure to a concentration of aCompound of the Invention such as Compound 1, particles, polymorphsand/or purified forms thereof of at least 1.5 μM for a defined period oftime, preferably at least 2 hours and the drug has to be substantiallycleared within 24 hours. Longer exposure to the compound can lead totoxicity and/or loss of selectivity.

To achieve this desired SPP or PPP, a Compound of the Invention can beadministered in a dose to achieve a blood concentration in a subject,e.g., a patient, of compound in the range of from at least about 0.02 μMto about 30 μM. For example, a Compound of the Invention can beadministered in a dose to achieve a blood concentration in a subject ofcompound at least about above 0.5 μM for a time of at least 2 hours, butless than 24 hours. For example, a Compound of the Invention can beadministered in a dose to achieve a blood concentration in a subject ofcompound at least about 2 μM for a time of at least 2 hours, but lessthan 24 hours.

Preferably, cancer cells must be exposed to a Compound of the Inventionsuch as Compound 1, particles, polymorphs and/or purified forms thereoffor 4 hours at concentration greater than 0.2 μM in order to inducecancer cell death. However, prolonged exposure does not contributesignificantly to the efficacy of a Compound of the Invention such asCompound 1, particles, polymorphs and/or purified forms thereof inkilling cancer cells. Compound 1 exhibited selective activity in killingcancer cells and sparing normal cells when the concentration of Compound1 was maintained at greater than from about 0.5 to about 3 μM for lessthan 24 hours. A reduced particle size of Compound 1 achieved thispreferred pharmacokinetic pattern and selective activity.

The selective activity of Compound 1 in killing cancer cells and sparingnormal cells is represented by the data in Table 14 and illustrated inFIG. 24. Exposure of cancer cells to Compound 1 at concentrations ofabout or above 0.2 and 30 μM for from about 4 hours up to about 24 hoursshowed selective killing of cancer cells and sparing of normal cells.Continuous exposure at these concentrations for durations of greaterthan 24 hours resulted in a loss of selectivity, in that normal cellswere also damaged. Exposure to Compound 1 at blood concentrations ofless than 0.5 μM resulted in no killing of cancer cells regardless ofthe amount of exposure time.

The dosing regimens described herein exhibit this preferred PK pattern.For example, the PK exhibited in patients receiving 500 mg BID inpatients is this preferred PK exposure pattern (FIG. 20) which showssustained exposure of Compound 1 above the therapeutic levels withsubstantial clearance of the drug by 24 hours. From 80 mg BID to 200 mgBID, SPP or PPP was achieved in patients with plasma drug concentrationincrease dose dependently. At 300 mg BID and 400 mg BID, it appearedplasma drug concentration appeared to have limited further increase over200 mg BID. However, it was found that 500 mg BID can surprisingly helpreduce inter-patient variation, namely all treated patients can achieveSPP with sufficiently high plasma drug concentration (FIG. 12). Finally,patients having exposure of Compound 1 above 1.6 uM for at least 4 hoursshow an improvement in progression free survival showing that thisexposure pattern leads to improved efficacy in humans. PK exposure ofCompound 1 above 1 uM correlates with an increase in progression freesurvival (FIG. 23) in patients with diverse cancers includingcolorectal, gastric, head and neck, melanoma, chondrosarcoma, lung,prostate, ovarian, adrenocorticoid and angiosarcoma. These data are verydifferent than what one would expect from preclinical experiments. Inpreclinical studies, Compound showed to kill cancer cells or cancer stemcells with IC₅₀ at about 100 to 200 nM. However, it was observedclinically in patients that those concentrations are not associated withclinical activity. In contrast, plasma concentration has to reach above1 uM to have signs of activity. Further increase of plasma drugconcentration to about or above 2 uM or 3 uM are associated withimproved signs of antitumor activity.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described.

What is claimed is:
 1. A composition comprising a purified form ofcompound 1, a salt, solvate, hydrate, or prodrug thereof,

wherein the composition has a purity greater than or equal to 95.0% asdetermined by high performance liquid chromatography (HPLC), nuclearmagnetic resonance (NMR) or both HPLC and NMR, and wherein thecomposition comprises less than or equal to 5% impurities.
 2. Thecomposition of claim 1, wherein the composition has a purity greaterthan or equal to 99% as determined by HPLC, NMR or both HPLC and NMR. 3.The composition of claim 1, wherein the composition comprises less thanor equal to 0.2% of each single impurity.
 4. The composition of claim 1,wherein the impurities comprise a residual solvent selected from thegroup consisting of ethyl acetate (EtOAc), toluene, ethanol, methanol,chloroform, and CH₂Cl₂/hexane.
 5. The composition of claim 1, whereinthe compound is in particle form and the particles comprise the compoundin a crystalline state.
 6. A compound having the structure shown below,a salt, solvate, hydrate, or prodrug thereof,

wherein the compound is in a particle form, and wherein the particle hasa diameter of less than or equal to about 20 micrometers.
 7. Apharmaceutical composition comprising a population of particles of thecompound of claim 6, wherein a fraction of the cumulative total of theparticles have a diameter in the range of 0.2 μm to 20 μm.
 8. Apharmaceutical composition comprising a population of particles of thecompound of claim 6, wherein 50% of the cumulative total of theparticles (D50) have a diameter of about 2 μm.
 9. The pharmaceuticalcomposition of claim 8, further comprising a pharmaceutically acceptableexcipient.
 10. A method of treating a cancer in a human subject, themethod comprising administering to a human subject in need thereof atherapeutically effective amount of the compound according to claim 1,or a pharmaceutically acceptable salt thereof.
 11. The method of claim10, wherein the cancer is selected from the group consisting ofcolorectal adenocarcinoma, breast cancer, ovarian cancer, head and neckcancer, melanoma, angiosarcoma, gastric adenocarcinoma, lung cancer,refractory cancer, recurrent cancer, metastatic cancer, andadrenocorticoid.
 12. The method of claim 10, wherein the cancer isassociated with overexpression of STAT3.
 13. A method of prolongingprogression free survival (PFS) in a cancer patient, the methodcomprising administering to a human subject in need thereof atherapeutically effective amount of the compound according to claim 1,or a pharmaceutically acceptable salt thereof.
 14. The method of claim13, wherein the cancer is selected from the group consisting ofcolorectal adenocarcinoma, breast cancer, ovarian cancer, head and neckcancer, melanoma, angiosarcoma, gastric adenocarcinoma, lung cancer,refractory cancer, recurrent cancer, metastatic cancer, andadrenocorticoid.
 15. The method of claim 13, wherein the cancer isassociated with overexpression of STAT3.